T cell recruiting polypeptides based on cd3 reactivity

ABSTRACT

T cell recruiting polypeptides are provided that bind CD3 on a T cell. The polypeptides can be used in methods for treatment of cancers.

FIELD OF THE INVENTION

The present invention provides multispecific T cell recruitingpolypeptides binding CD3 on a T cell and at least one antigen on atarget cell. The present invention also relates to the monovalent T cellrecruiting polypeptides for use in these multispecific polypeptides. Theinvention also provides methods for treatment and kits providing thesame.

BACKGROUND

Cancer takes an enormous human toll around the world. It is nowadays theworld's leading cause of death, followed by heart disease and stroke.Cancers figure among the leading causes of morbidity and mortalityworldwide, with approximately 14 million new cases and 8.2 millioncancer related deaths in 2012. The number of new cases is expected torise by about 70% over the next 2 decades (source: WHO Cancer). Thetotal economic impact of premature death and disability from cancerworldwide was about $900 billion in 2008, representing 1.5% of theworld's gross domestic product.

Available treatment regimens for solid tumours typically include acombination of surgical resection chemotherapy and radiotherapy. In 40years of clinical experience little progress has been achieved,especially in advanced stages of cancer.

New therapies combatting cancer are eagerly awaited.

Antibody therapy is now an important part of the physician'sarmamentarium to battle diseases and especially cancer. Monoclonalantibodies have been established as a key therapeutic approach for arange of diseases already for several years. All of thecontemporaneously approved antibody therapies rely on monospecificmonoclonal antibodies (mAbs). Until today, most of the targets of themAbs require either an agonistic or an antagonistic approach. Whereastargeting of cell-surface antigens themselves can mediate antitumoractivity through the induction of apoptosis, most mAb-based activityagainst hematologic malignancies is reliant on either Fc-mediatedeffector functions such as complement dependent cytotoxicity (CDC) andantibody-dependent cell-mediated cytotoxicity (ADCC).

Immunotherapy has emerged as a rapidly growing area of cancer research.Immunotherapy is directing the body's immune surveillance system, and inparticular T cells, to cancer cells.

Cytotoxic T cells (CTL) are T lymphocytes that kill cancer cells, cellsthat are infected (particularly with viruses), or cells that are damagedin other ways. T lymphocytes (or T cells) express the T cell receptor orTCR molecule and the CD3 receptor on the cell surface. The αβ TCR-CD3complex (or “TCR complex”) is composed of six different type Isingle-spanning transmembrane proteins: the TCRα and TCRβ chains thatform the TCR heterodimer responsible for ligand recognition, and thenon-covalently associated CD3γ, CD3δ, CD3ε and ζ chains, which bearcytoplasmic sequence motifs that are phosphorylated upon receptoractivation and recruit a large number of signalling components (Call etal. 2004, Molecular Immunology 40: 1295-1305).

Both α and β chains of the T cell receptor consist of a constant domainand a variable domain. Physiologically, the αβ chains of the T cellreceptor recognize the peptide loaded MHC complex and couple uponengagement to the CD3 chains. These CD3 chains subsequently transducethe engagement signal to the intracellular environment.

Considering the potential of naturally occurring Cytotoxic T lymphocytes(CTLs) to mediate cell lysis, various strategies have been explored torecruit CTLs to mediate tumour cell killing. Since T lymphocytes lackthe expression of Fc receptors, they are not recruited to a tumour sitethrough the Fc tail of an anti-tumour monoclonal. As an alternative, thepatient's T cells were modified with a second TCR of known specificityfor a defined tumour antigen. This adoptive cell transfer is by naturehighly personalized and labour intensive. However, the main problem of Tcell therapies remains the large number of immune escape mechanisms knowto occur in cancer patients (Nagorsen et al. 2012, Pharmacology &Therapeutics 136: 334-342).

Rather than eliciting specific T cell responses, which rely onexpression by cancer cells of MHC molecules and the presence,generation, transport and display of specific peptide antigens, morerecent developments have attempted to combine the advantages ofimmunotherapy with antibody therapy by engaging all cytotoxic T cells ofa patient in a polyclonal fashion via recombinant antibody basedtechnologies: “bispecifics”.

Bispecific antibodies have been engineered that have a tumourrecognition part on the one arm (target-binding arm) whereas the otherarm of the molecule has specificity for a T cell antigen(effector-binding arm), mostly CD3. Through the simultaneous binding ofthe two arms to their respective target antigens, T lymphocytes aredirected towards and activated at the tumour cell where they can exerttheir cytolytic function.

The concept of using bispecific antibodies to activate T cells againsttumour cells was described more than 20 years ago, but manufacturingproblems and clinical failures sent the field into stagnation. Smallerformat bispecifics were developed, which more easily penetrate tissuesand tumours than conventional antibodies. In addition, the smallerformat is better at creating the cytotoxic synapses, which kill thetarget cell. It was thought that the smaller format bispecifics would beeasier to manufacture and less immunogenic than conventional antibodies.However, the smaller bispecific BiTE molecules, consisting of two singlechain variable fragments (scFvs) joined by a 5 amino acid peptidelinker, present a lack of stability (scFvs tend to aggregate), lowexpression titres and poor solubility. Moreover, the first clinicaltrials of Blinatumomab (a BITE molecule), which recognizes CD3 chains,were prematurely stopped due to neurologic adverse events, cytokinerelease syndrome and infections on the one hand and the absence ofobjective clinical responses or robust signs of biological activity onthe other hand. Efficacy aside, BiTEs must be continuouslyinfused—probably due to the lack of an Fc domain—which does notcontribute to a patient compliance. The same problem holds true forDARTs (dual affinity retargeting molecules developed by MacroGenics), inwhich the heavy variable domain from one antibody (Ab) is linked withthe light variable domain of another Ab. MacroGenics now attempts tosolve this problem by fusing an Fc domain onto its next generationDARTs, which makes the molecule not only bigger, but also results inmanufacturing problems and importation of other Fc functions. The largerformat with Fc will have a better PK, but re-introduces the risk ofoff-target activity. (Garber 2014, Nature reviews 13: 799-801) Thereremains the need for alternative bispecific formats.

SUMMARY OF THE INVENTION

The invention solves this problem by providing multispecificpolypeptides comprising a first and at least one further immunoglobulinsingle variable domain (ISV), wherein said first ISV has high affinityfor/binds to CD3; said at least one further ISV has high affinityfor/binds to an antigen present on a target cell. In a particularaspect, the binding of the first ISV will activate the inherentcytolytic potential of the T cell against the target cell independentlyof MHC1.

Thus, in a first aspect the present invention provides a polypeptidecomprising a first and a second immunoglobulin single variable domain(iSV), wherein

-   -   said first ISV has high affinity for/binds to cluster of        differentiation 3 (CD3) present on a T cell;    -   said second ISV has high affinity for/binds to a first antigen        on a target cell;    -   wherein said first antigen is different from said CD3; and    -   wherein said target cell is different from said T cell.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said polypeptide directs the T-cell to thetarget cell.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said polypeptide induces T cell activation.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said T cell activation is independent from MHCrecognition.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said T cell activation depends on presentingsaid polypeptide bound to said first antigen on a target cell to a Tcell.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said T cell activation causes one or morecellular response of said T cell, wherein said cellular response isselected from the group consisting of proliferation, differentiation,cytokine secretion, cytotoxic effector molecule release, cytotoxicactivity, expression of activation markers and redirected target celllysis.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said T cell activation causes inhibition of anactivity of said target cell by more than about 10%, such as 20%, 30%,or 40% or even more than 50%, such as more than 60%, such as 70%, 80%,or even more than 90%, such as 100%.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said first ISV binds to CD3γ (SEQ ID NO: 292),to CD3δ (SEQ ID NO: 291) and/or CD3ε (SEQ ID NO: 293) of the TCRcomplex, or polymorphic variants or isoforms thereof.

Alternatively, the present invention provides a polypeptide as describedherein, wherein said first ISV binds to CD3γ (SEQ ID NO: 379), to CD3δ(SEQ ID NO: 291) and/or CD3ε (SEQ ID NO: 380) of the TCR complex, orpolymorphic variants or isoforms thereof.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said polypeptide and/or first ISV has an onrate constant (Kon) for binding to said CD3 selected from the groupconsisting of at least about 10² M⁻¹s⁻¹, at least about 10³ M⁻¹s⁻¹, atleast about 10⁴ M⁻¹s⁻¹, at least about 10⁵ M⁻¹s⁻¹, at least about 10⁶M⁻¹s⁻¹, 10⁷ M⁻¹s⁻¹, at least about 10⁸ M⁻¹s⁻¹, at least about 10⁸M⁻¹s⁻¹, and at least about 10¹⁶ M⁻¹s⁻¹, preferably as measured bysurface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said polypeptide and/or first ISV has an offrate constant (Koff) for binding to said CD3 selected from the groupconsisting of at most about 10⁻³s⁻¹, at most about 10⁻⁴s⁻¹, at mostabout 10⁻⁵s⁻¹, at most about 10⁻⁶s⁻¹, at most about 10⁻⁷s¹, at mostabout 10⁸s⁻¹, at most about and at most about 10¹⁰s⁻¹, preferably asmeasured by surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said first ISV binds to said CD3 with an EC50value of between 100 nM and 1 pM, such as at an average EC50 value of100 nM or less, even more preferably at an average EC50 value of 90 nMor less, such as less than 80, 70, 60, 50, 40, 30, 20, 10, 5 nM or evenless, such as less than 4, 3, 2, or 1 nM or even less, such as less than500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 pM, oreven less, such as less than 4 pM, preferably as measured by flowcytometry.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said first ISV binds to said CD3 with anaverage KD value of between 100 nM and 10 pM, such as at an average KDvalue of 90 nM or less, even more preferably at an average KD value of80 nM or less, such as less than 70, 60, 50, 40, 30, 20, 10, 5 nM oreven less, such as less than 4, 3, 2, or 1 nM, such as less than 500,400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 pM, or even lesssuch, as less than 10 pM. Preferably, the KID is determined by SPR, forinstance as determined by Proteon.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 81-100; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 81; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 101-122; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 101; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 123-143; and        -   (f) amino acid sequences that have 3, 2, or 1 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 123.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

(i) CDR1 is chosen from the group consisting of:

-   -   (a) SEQ ID NOs: 81-100; or (b) amino acid sequences that have 4,        3, 2, or 1 amino acid(s) difference with the amino acid sequence        of SEQ ID NO: 81 or with any of SEQ ID NOs: 81-100, provided        that the polypeptide comprising the CDR1 with 4, 3, 2, or 1        amino acid(s) difference binds CD3 with about the same or a        higher affinity compared to the binding by the polypeptide        comprising the CDR1 without the 4, 3, 2, or 1 amino acid(s)        difference, said affinity as measured by surface plasmon        resonance; and/or

(ii) CDR2 is chosen from the group consisting of:

-   -   (c) SEQ ID NOs: 101-122; or (d) amino acid sequences that have        4, 3, 2, or 1 amino acid(s) difference with the amino acid        sequence of SEQ ID NO: 101 or with any of SEQ ID NOs: 101-122,        provided that the polypeptide comprising the CDR2 with 4, 3, 2,        or 1 amino acid(s) difference binds CD3 with about the same or a        higher affinity compared to the binding by the polypeptide        comprising the CDR2 without the 4, 3, 2, or 1 amino acid(s)        difference, said affinity as measured by surface plasmon        resonance; and/or

(iii) CDR3 is chosen from the group consisting of:

-   -   (e) SEQ ID NOs: 123-143; or (f) amino acid sequences that have        4, 3, 2, or 1 amino acid(s) difference with the amino acid        sequence of SEQ ID NO: 123 or with any of SEQ ID NOs: 123-143,        provided that the polypeptide comprising the CDR3 with 4, 3, 2,        or 1 amino acid(s) difference binds CD3 with about the same or a        higher affinity compared to the binding by the polypeptide        comprising the CDR3 without the 4, 3, 2, or 1 amino acid(s)        difference, said affinity as measured by surface plasmon        resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which CDR1 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 81; and    -   (b) amino acid sequences that have 1 or 2 amino acid        difference(s) with SEQ ID NO: 81, wherein        -   at position 1 the G has been changed into R;        -   at position 3 the T has been changed into A;        -   at position 4 the Y has been changed into F;        -   at position 8 the S has been changed into G; and/or        -   at position 10 the G has been changed into A.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which CDR2 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 101; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NO: 101, wherein        -   at position 3 the V has been changed into T or A;        -   at position 5 the S has been changed into T;        -   at position 6 the G has been changed into D or E; and/or        -   at position 9 the T has been changed into S, A or P.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which CDR3 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 123; and    -   (b) amino acid sequences that have 1 or 2 amino acid        difference(s) with SEQ ID NO: 123, wherein        -   at position 2 the I has been changed into T;        -   at position 9 the I has been changed into V; and/or        -   at position 10 the A has been changed into P.

Preferably, the polypeptide comprising the one or more CDRs with 3, 2,or 1 amino acid(s) difference binds CD3 with about the same or a higheraffinity compared to the binding by the polypeptide comprising the CDRswithout the 3, 2, or 1 amino acid(s) difference, said affinity asmeasured by surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

(i) CDR1 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 81; and    -   (b) amino acid sequences that have 1 or 2 amino acid        difference(s) with SEQ ID NO: 81, wherein    -   at position 1 the G has been changed into R;        -   at position 3 the T has been changed into A;        -   at position 4 the Y has been changed into F;        -   at position 8 the S has been changed into G; and/or        -   at position 10 the G has been changed into A,    -    provided that the polypeptide comprising the CDR1 with 2, or 1        amino acid(s) difference binds CD3 with about the same or a        higher affinity compared to the binding by the polypeptide        comprising the CDR1 without the 2, or 1 amino acid(s)        difference, said affinity as measured by surface plasmon        resonance;

and in which

(ii) CDR2 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 101; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NO: 101, wherein        -   at position 3 the V has been changed into T or A;        -   at position 5 the S has been changed into T;        -   at position 6 the G has been changed into D or E; and/or        -   at position 9 the T has been changed into S, A or P,    -    provided that the polypeptide comprising the CDR2 with 3, 2, or        1 amino acid(s) difference binds CD3 with about the same or a        higher affinity compared to the binding by the polypeptide        comprising the CDR2 without the 3, 2, or 1 amino acid(s)        difference, said affinity as measured by surface plasmon        resonance;

and in which

(iii) CDR3 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 123; and    -   (b) amino acid sequences that have 1, or 2 amino acid        difference(s) with SEQ ID NO: 123, wherein        -   at position 2 the I has been changed into T;        -   at position 9 the I has been changed into V; and/or        -   at position 10 the A has been changed into P,    -    provided that the polypeptide comprising the CDR3 with 2, or 1        amino acid(s) difference binds CD3 with about the same or a        higher affinity compared to the binding by the polypeptide        comprising the CDR3 without the 2, or 1 amino acid(s)        difference, said affinity as measured by surface plasmon        resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 81-87; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 81; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 101-109; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 101; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 123-127; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 123.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 81-87; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 81, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR′ without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 101-109; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 101, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 123-127; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 123, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR1 isrepresented by SEQ ID NO: 81, CDR2 is represented by SEQ ID NO: 101, andCDR3 is represented by SEQ ID NO: 123.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV is chosen from the groupconsisting of SEQ ID NOs: 1-50.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV cross-blocks the binding toCD3 by at least one of the polypeptides with SEQ ID NOs: 1-50.

In a further aspect, the present invention provides a polypeptide asdescribed herein in which said first ISV is cross-blocked from bindingto CD3 by at least one of the polypeptides with SEQ ID NOs: 1-50.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR1 is SEQID NO: 88.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR2 is SEQID NO: 110.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR3 is SEQID NO: 128.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NO: 88; and        -   (b) amino acid sequences that have 1, 2, 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 88; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NO: 110; and        -   (d) amino acid sequences that have 1, 2, 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 110; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NO: 128; and        -   (f) amino acid sequences that have 1, 2, 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 128.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 88; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 88, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 110; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 110, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 128; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 128, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR1 isrepresented by SEQ ID NO: 88, CDR2 is represented by SEQ ID NO: 110, andCDR3 is represented by SEQ ID NO: 128.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV is SEQ ID NOs: 51.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV cross-blocks the binding toCD3 by the polypeptide with SEQ ID NOs: 51.

In a further aspect, the present invention provides a polypeptide asdescribed herein in which said first ISV is cross-blocked from bindingto CD3 by the polypeptide with SEQ ID NOs: 51.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR1 is SEQID NO: 90.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which CDR2 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 112; and    -   (b) amino acid sequence that has 1 amino acid difference with        SEQ ID NO: 112, wherein        -   at position 2 the V has been changed into A.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR3 is SEQID NO: 130.

Preferably, the polypeptide comprising the one or more CDRs with 1 aminoacid difference binds CD3 with about the same or a higher affinitycompared to the binding by the polypeptide comprising the CDRs withoutthe 1 amino acid difference, said affinity as measured by surfaceplasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

(i) CDR1 is SEQ ID NO: 90; and

and in which

(ii) CDR2 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 112; and    -   (b) amino acid sequences that have 1 amino acid difference with        SEQ ID NO: 112, wherein        -   at position 2 the V has been changed into A,    -    provided that the polypeptide comprising the CDR2 with 1 amino        acid difference binds CD3 with about the same or a higher        affinity compared to the binding by the polypeptide comprising        the CDR2 without the 1 amino acid difference, said affinity as        measured by surface plasmon resonance;

and in which

(iii) CDR3 is SEQ ID NO: 130.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NO: 90; and        -   (b) amino acid sequences that have 1, 2, 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 90; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 112-113; and        -   (d) amino acid sequences that have 1, 2, 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 112; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NO: 130; and        -   (f) amino acid sequences that have 1, 2, 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 130.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 90; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 90, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 112-113; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 112, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 130; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 130, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR1 isrepresented by SEQ ID NO: 90, CDR2 is represented by SEQ ID NO: 112, andCDR3 is represented by SEQ ID NO: 130.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV is chosen from the groupconsisting of SEQ ID NOs: 53-56.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV cross-blocks the binding toCD3 by at least one of the polypeptides with SEQ ID NOs: 53-56.

In a further aspect, the present invention provides a polypeptide asdescribed herein in which said first ISV is cross-blocked from bindingto CD3 by at least one of the polypeptides with SEQ ID NOs: 53-56.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR1 is SEQID NO: 89.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR2 is SEQID NO: 111.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR3 is SEQID NO: 129.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NO: 89; and        -   (b) amino acid sequences that have 1, 2, 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 89; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NO: 111; and        -   (d) amino acid sequences that have 1, 2, 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 111; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NO: 129; and        -   (f) amino acid sequences that have 1, 2 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 129.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 89; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 89, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 111; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 111, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 129; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 129, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR1 isrepresented by SEQ ID NO: 89, CDR2 is represented by SEQ ID NO: 111, andCDR3 is represented by SEQ ID NO: 129.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV is SEQ ID NOs: 52.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV cross-blocks the binding toCD3 by the polypeptide with SEQ ID NOs: 52.

In a further aspect, the present invention provides a polypeptide asdescribed herein in which said first ISV is cross-blocked from bindingto CD3 by the polypeptides with SEQ ID NOs: 52.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which CDR1 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 91; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid(s)        difference with SEQ ID NO: 91, wherein        -   at position 6 the R has been changed into N or T;        -   at position 7 the N has been changed into H; and/or        -   at position 8 the M has been changed into T.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which CDR2 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 114; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid(s)        difference with SEQ ID NO: 114, wherein        -   at position 1 the R has been changed into Q;        -   at position 3 the T has been changed into S; and/or        -   at position 7 the D has been changed into A or K.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which CDR3 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 131; and    -   (b) amino acid sequences that have 1 amino acid difference with        SEQ ID NO: 131, wherein        -   at position 2 the S has been changed into R; and/or        -   at position 6 the S has been changed into V.

Preferably, the polypeptide comprising the one or more CDRs with 3, 2,or 1 amino acid(s) difference binds CD3 with about the same or a higheraffinity compared to the binding by the polypeptide comprising the CDRswithout the 3, 2, or 1 amino acid(s) difference, said affinity asmeasured by surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

(i) CDR1 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 91; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NO: 91, wherein        -   at position 6 the R has been changed into N or T;        -   at position 7 the N has been changed into H; and/or        -   at position 8 the M has been changed into T,    -    provided that the polypeptide comprising the CDR1 with 3, 2, or        1 amino acid(s) difference binds CD3 with about the same or a        higher affinity compared to the binding by the polypeptide        comprising the CDR1 without the 3, 2, or 1 amino acid(s)        difference, said affinity as measured by surface plasmon        resonance;

and in which

(ii) CDR2 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 114; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NO: 114, wherein        -   at position 1 the R has been changed into Q;        -   at position 3 the T has been changed into S; and/or        -   at position 7 the D has been changed into A or K,    -    provided that the polypeptide comprising the CDR2 with 3, 2, or        1 amino acid(s) difference binds CD3 with about the same or a        higher affinity compared to the binding by the polypeptide        comprising the CDR2 without the 3, 2, or 1 amino acid(s)        difference, said affinity as measured by surface plasmon        resonance;

and in which

(iii) CDR3 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 131; and    -   (b) amino acid sequences that have 1 amino acid difference with        SEQ ID NO: 131, wherein        -   at position 2 the S has been changed into R; and/or        -   at position 6 the S has been changed into V,    -    provided that the polypeptide comprising the CDR3 with 1 amino        acid difference binds CD3 with about the same or a higher        affinity compared to the binding by the polypeptide comprising        the CDR3 without the 1 amino acid difference, said affinity as        measured by surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to F114, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 91-93; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 91; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 114-117; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 114; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 131-133; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 131.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 91-93; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 91, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 114-117; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 114, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or        -   (iii) CDR3 is chosen from the group consisting of:    -   (e) SEQ ID NOs: 131-133; and    -   (f) amino acid sequences that have 4, 3, 2, or 1 amino acid(s)        difference with the amino acid sequence of SEQ ID NO: 131,        provided that the polypeptide comprising the CDR3 with 4, 3, 2,        or 1 amino acid(s) difference binds CD3 with about the same or a        higher affinity compared to the binding by the polypeptide        comprising the CDR3 without the 4, 3, 2, or 1 amino acid(s)        difference, said affinity as measured by surface plasmon        resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR1 isrepresented by SEQ ID NO: 91, CDR2 is represented by SEQ ID NO: 114, andCDR3 is represented by SEQ ID NO: 131.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV is chosen from the groupconsisting of SEQ ID NOs: 57-65.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV cross-blocks the binding toCD3 by at least one of the polypeptides with SEQ ID NOs: 57-65.

In a further aspect, the present invention provides a polypeptide asdescribed herein in which said first ISV is cross-blocked from bindingto CD3 by at least one of the polypeptides with SEQ ID NOs: 57-65.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which CDR1 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 94; and    -   (b) amino acid sequences that have 1, 2, 3 or 4 amino acid(s)        difference with SEQ ID NO: 94, wherein        -   at position 3 the S has been changed into T, A or G;        -   at position 5 the N has been changed into S;        -   at position 6 the M has been changed into T or A; and/or        -   at position 9 the L has been changed into M.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which CDR2 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 118; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid(s)        difference with SEQ ID NO: 118, wherein        -   at position 2 the H has been changed into V;        -   at position 5 the S has been changed into H or A;        -   at position 8 the N has been changed into S; and/or        -   at position 10 the Y has been changed into F.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first iSV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which CDR3 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 134; and    -   (b) amino acid sequences that have 1, 2, 3, 4 or 5 amino acid(s)        difference with SEQ ID NO: 134, wherein        -   at position 6 the A has been changed into S or 0;        -   at position 7 the F has been changed into Y or A;        -   at position 8 the R has been changed into H;        -   at position 9 the S has been changed into A;        -   at position 11 the G has been changed into D, T, N, S, k or            R; and/or        -   at position 14 the V has been changed into I.

Preferably, the polypeptide comprising the one or more CDRs with 5, 4,3, 2, or 1 amino acid(s) difference binds CD3 with about the same or ahigher affinity compared to the binding by the polypeptide comprisingthe CDRs without the 5, 4, 3, 2, or 1 amino acid(s) difference, saidaffinity as measured by surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which

(i) CDR1 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 94; and    -   (b) amino acid sequences that have 1, 2, 3 or 4 amino acid        difference(s) with SEQ ID NO: 94, wherein        -   at position 3 the S has been changed into T, A or G;        -   at position 5 the N has been changed into S;        -   at position 6 the M has been changed into T or A; and/or        -   at position 9 the L has been changed into M,    -    provided that the polypeptide comprising the CDR1 with 4, 3, 2,        or 1 amino acid(s) difference binds CD3 with about the same or a        higher affinity compared to the binding by the polypeptide        comprising the CDR1 without the 4, 3, 2, or 1 amino acid(s)        difference, said affinity as measured by surface plasmon        resonance;

and in which

(ii) CDR2 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 118; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NO: 118, wherein        -   at position 2 the H has been changed into V;        -   at position 5 the S has been changed into H or A;        -   at position 8 the N has been changed into S; and/or        -   at position 10 the Y has been changed into F,    -    provided that the polypeptide comprising the CDR2 with 3, 2, or        1 amino acid(s) difference binds CD3 with about the same or a        higher affinity compared to the binding by the polypeptide        comprising the CDR2 without the 3, 2, or 1 amino acid(s)        difference, said affinity as measured by surface plasmon        resonance;

and in which

(iii) CDR3 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 134; and    -   (b) amino acid sequences that have 1, 2, 3, 4 or 5 amino acid        difference(s) with SEQ ID NO: 134, wherein        -   at position 6 the A has been changed into S or 0;        -   at position 7 the F has been changed into Y or A;        -   at position 8 the R has been changed into H;        -   at position 9 the S has been changed into A;        -   at position 11 the G has been changed into D, T, N, S, K or            R; and/or        -   at position 14 the V has been changed into I,    -    provided that the polypeptide comprising the CDR3 with 5, 4, 3,        2, or 1 amino acid(s) difference binds CD3 with about the same        or a higher affinity compared to the binding by the polypeptide        comprising the CDR3 without the 5, 4, 3, 2, or 1 amino acid(s)        difference, said affinity as measured by surface plasmon        resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 94-100; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 94; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 118-122; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 118; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 134-143; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 134.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 94-100; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 94, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 118-122; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 118, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 134-143; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 134, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR1 isrepresented by SEQ ID NO: 94, CDR2 is represented by SEQ ID NO: 118, andCDR3 is represented by SEQ ID NO: 134.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV is chosen from the groupconsisting of SEQ ID NOs: 66-80.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which said first ISV cross-blocks the binding toCD3 by at least one of the polypeptides with SEQ ID NOs: 66-80.

In a further aspect, the present invention provides a polypeptide asdescribed herein in which said first ISV is cross-blocked from bindingto CD3 by at least one of the polypeptides with SEQ ID NOs: 66-80.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said first antigen on a target cell is atumour antigen, preferably a tumour associated antigen (TAA).

In a further aspect, the present invention provides a polypeptide asdescribed herein, further comprising a third ISV, which has highaffinity for/binds to a second antigen on a target cell, wherein saidsecond antigen is different from said first antigen.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said second antigen on a target cell is atumour antigen, preferably a tumour associated antigen (TAA).

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said first antigen and said second antigen arepresent on the same target cells.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said first antigen and said second antigen arepresent on different target cells.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said TAA's are independently chosen from thegroup consisting of Melanoma-associated Chondroitin Sulfate Proteoglycan(MCSP), Epidermal Growth Factor Receptor (EGFR), Fibroblast ActivationProtein (FAP), MART-1, carcinoembryonic antigen (CEA), gp100, MAGE-1,HER-2, Lewis^(Y) antigens, CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4,Tim-3, CD25, TAG-72, Ep-CAM, PSMA, PSA, GD2, GD3, CD4, CD5, CD19, CD20,CD22, CD33, CD36, CD45, CD52, CD147, growth factor receptors includingErbB3 and ErbB4, Cytokine receptors including Interleukin-2 receptorgamma chain (CD132 antigen), Interleukin-10 receptor alpha chain(IL-10R-A), Interleukin-10 receptor beta chain (IL-10R-B),Interleukin-12 receptor beta-1 chain (IL-12R-beta1), Interleukin-12receptor beta-2 chain (IL-12 receptor beta-2), Interleukin-13 receptoralpha-1 chain (IL-13R-alpha-1) (CD213a1 antigen), Interleukin-13receptor alpha-2 chain (Interleukin-13 binding protein), Interleukin-17receptor (11-17 receptor), Interleukin-17B receptor (IL-17B receptor),Interleukin 21 receptor precursor (IL-21R), Interleukin-1 receptor typeI (IL-1R-1) (CD121a), Interleukin-1 receptor type II (IL-1R-beta)(CDw121b), Interleukin-1 receptor antagonist protein (IL-1ra),Interleukin-2 receptor alpha chain (CD25 antigen), Interleukin-2receptor beta chain (CD122 antigen), Interleukin-3 receptor alpha chain(IL-3R-alpha) (CD123 antigen), CD30, 1123R, IGF-1R, IL5R, IgE, CD248(endosialin), CD44v6, gpA33, Ron, Trop2, PSCA, claudin 6, claudin 18.2,CLEC12A, CD38, ephA2, c-Met, CD56, MUC16, EGFRvIII, AGS-16, CD27L,Nectin-4, SLITRK6, mesothelin, folate receptor, tissue factor, axl,glypican-3, CA9, Cripto, CD138, CD37, MUC1, CD70, gastrin releasingpeptide receptor, PAP, CEACAM5, CEACAM6, CXCR7, N-cadherin, FXYD2 gammaa, CD21, CD133, Na/K-ATPase, mIgM (membrane-bound IgM), mIgA(membrane-bound IgA), Mer, Tyro2, CD120, CD95, CA 195, DR5, DR6, DcR3and CAIX, including related polymorphic variants and isoforms.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said TAA is CD20 (UniProt 11836), HER2(Uniprot P04626), polymorphic variants or isoforms thereof.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said first antigen and said second antigen arechosen from the group consisting of:

-   -   EGFR as a first antigen and CEA as a second antigen;    -   CD19 as a first antigen and CD20 as a second antigen;    -   CD19 as a first antigen and CD22 as a second antigen;    -   CD123 as a first antigen and Tim-3 as a second antigen; and    -   CD132 as a first antigen and CD69 as a second antigen.

In a further aspect, the present invention provides a polypeptide asdescribed herein, further comprising a serum protein binding moiety.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said serum protein binding moiety binds serumalbumin.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said serum protein binding moiety is an ISVbinding serum albumin.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said ISV binding serum albumin essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich CDR1 is SFGMS (SEQ ID NO: 373), CDR2 is SISGSGSDTLYADSVKG (SEQ IDNO: 374) and CDR3 is GGSLSR (SEQ ID NO: 375), CDR determined accordingto Kabat definition; and/or in which CDR1 is GFTFSSFGMS (SEQ ID NO: 376)or GFTFRSFGMS (SEQ ID NO: 377), CDR2 is SISGSGSDTL (SEQ ID NO: 378) andCDR3 is GGSLSR (SEQ ID NO: 375), CDR determined according to Kontermann2010.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said ISV binding serum albumin is selectedfrom Alb8, Alb23, Alb129, Alb132, Alb11, Alb11 (S112K)-A, Alb82,Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, and Alb82-GGG (SEQ IDNOs: 348 to 360).

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said ISVs are directly linked to each other orare linked via a linker.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said first ISV and/or said second ISV and/orpossibly said third ISV and/or possibly said ISV binding serum albuminare linked via a linker.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said linker is chosen from the groupconsisting of linkers of 5GS, 7GS, 9G5, 10GS, 15GS, 18GS, 20GS, 25GS,30GS and 35GS (SEQ ID NOs: 362 to 372).

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said serum protein binding moiety is anon-antibody based polypeptide.

In a further aspect, the present invention provides a polypeptide asdescribed herein, further comprising PEG.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said ISV is a Nanobody®*, a V_(HH), ahumanized V_(HH), or a camelized V_(H).

In a further aspect, the present invention provides a polypeptidewherein said first ISV is chosen from the group consisting of SEQ IDNOs: 1 to 80.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said first ISV is chosen from the groupconsisting of SEQ ID NOs: 1 to 80, and wherein said second ISV is chosenfrom the group consisting of SEQ ID NOs: 297 to 304.

In a further aspect, the present invention provides a polypeptide chosenfrom the group consisting of SEQ ID NOs: 249-250, 252-253, 255-256,258-260, 263, 265-283, 286-289, 306-307, 309-310, 312-313, 315-317, 320,322-340 and 343-346.

In a further aspect, the present invention provides a polypeptide thatspecifically binds CD3 and that comprises or essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 81-100; or        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of any of            SEQ ID NOs: 81-100, provided that the polypeptide comprising            the CDR1 with 4, 3, 2, or 1 amino acid(s) difference binds            CD3 with about the same or a higher affinity compared to the            binding by the polypeptide comprising the CDR1 without the            4, 3, 2, or 1 amino acid(s) difference, said affinity as            measured by surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 101-122; or        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino add sequence of any of SEQ            ID NOs: 101-122, provided that the polypeptide comprising            the CDR2 with 4, 3, 2, or 1 amino acid(s) difference binds            CD3 with about the same or a higher affinity compared to the            binding by the polypeptide comprising the CDR2 without the            4, 3, 2, or 1 amino acid(s) difference, said affinity as            measured by surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 123-143; or        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of any of            SEQ ID NOs: 123-143, provided that the polypeptide            comprising the CDR3 with 4, 3, 2, or 1 amino acid(s)            difference binds CD3 with about the same or a higher            affinity compared to the binding by the polypeptide            comprising the CDR3 without the 4, 3, 2, or 1 amino acid(s)            difference, said affinity as measured by surface plasmon            resonance.

The present invention also provides a polypeptide as described herein,in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 81-87; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 81, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 101-109; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 101, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 123-127; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 123, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which CDR1 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 81; and    -   (b) amino acid sequences that have 1 or 2 amino acid        difference(s) with SEQ ID NO: 81, wherein        -   at position 1 the G has been changed into R;        -   at position 3 the T has been changed into A;        -   at position 4 the Y has been changed into F;        -   at position 8 the S has been changed into G; and/or        -   at position 10 the G has been changed into A.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which CDR2 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 101; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NO: 101, wherein        -   at position 3 the V has been changed into T or A;        -   at position 5 the S has been changed into T;        -   at position 6 the G has been changed into D or E; and/or        -   at position 9 the T has been changed into S, A or P.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which CDR3 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 123; and    -   (b) amino acid sequences that have 1 or 2 amino acid        difference(s) with SEQ ID NO: 123, wherein        -   at position 2 the I has been changed into T;        -   at position 9 the I has been changed into V; and/or        -   at position 10 the A has been changed into P.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR1 is represented by SEQ ID NO: 81, CDR2is represented by SEQ ID NO: 101, and CDR3 is represented by SEQ ID NO:123.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 88; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 88, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 110; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 110, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 128; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 128, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR1 is SEQ ID NO: 88.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR2 is SEQ ID NO: 110.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR3 is SEQ ID NO: 128.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR1 is represented by SEQ ID NO: 88, CDR2is represented by SEQ ID NO: 110, and CDR3 is represented by SEQ ID NO:128.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which:

-   -   (i) CDR1 is chosen from the group consisting of        -   (a) SEQ ID NO: 90; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 90, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 112-113; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 112, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NO: 130; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 130, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR1 is SEQ ID NO: 90.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR2 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 112; and    -   (b) amino acid sequence that has 1 amino acid difference with        SEQ ID NO: 112, wherein        -   at position 2 the V has been changed into A.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR3 is SEQ ID NO: 130.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR1 is represented by SEQ ID NO: 90, CDR2is represented by SEQ ID NO: 112, and CDR3 is represented by SEQ ID NO:130.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 89; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 89, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 111; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 111, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 129; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 129, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR1 is SEQ ID NO: 89.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR2 is SEQ ID NO: 111.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR3 is SEQ ID NO: 129.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR1 is represented by SEQ ID NO: 89, CDR2is represented by SEQ ID NO: 111, and CDR3 is represented by SEQ ID NO:129.

The present invention also provides a polypeptide as described herein,in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 91-93; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 91, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 114-117; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 114, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 131-133; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 131, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which CDR1 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 91; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid(s)        difference with SEQ ID NO: 91, wherein        -   at position 6 the R has been changed into N or T;        -   at position 7 the N has been changed into H; and/or        -   at position 8 the M has been changed into T.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which CDR2 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 114; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid(s)        difference with SEQ ID NO: 114, wherein        -   at position 1 the R has been changed into Q;        -   at position 3 the T has been changed into S; and/or        -   at position 7 the D has been changed into A or K.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which CDR3 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 131; and    -   (b) amino acid sequences that have 1 amino acid difference with        SEQ ID NO: 131, wherein        -   at position 2 the S has been changed into R; and/or        -   at position 6 the S has been changed into V.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR1 is represented by SEQ ID NO: 91, CDR2is represented by SEQ ID NO: 114, and CDR3 is represented by SEQ ID NO:131.

The present invention also provides a polypeptide as described herein,in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 94-100; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 94, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 118-122; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 118, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 134-143; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 134, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which CDR1 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 94; and    -   (b) amino acid sequences that have 1, 2, 3 or 4 amino acid(s)        difference with SEQ ID NO: 94, wherein        -   at position 3 the S has been changed into T, A or G;        -   at position 5 the N has been changed into S;        -   at position 6 the M has been changed into T or A; and/or        -   at position 9 the has been changed into M.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which CDR2 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 118; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid(s)        difference with SEQ ID NO: 118, wherein        -   at position 2 the H has been changed into V;        -   at position 5 the S has been changed into H or A;        -   at position 8 the N has been changed into S; and/or        -   at position 10 the Y has been changed into F.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which CDR3 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 134; and    -   (b) amino acid sequences that have 1, 2, 3, 4 or 5 amino acid(s)        difference with SEQ ID NO: 134, wherein        -   at position 6 the A has been changed into S or D;        -   at position 7 the F has been changed into Y or A;        -   at position 8 the R has been changed into H;        -   at position 9 the S has been changed into A;        -   at position 11 the G has been changed into D, T, N, S, K or            R; and/or        -   at position 14 the V has been changed into I.

In a further aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR1 is represented by SEQ ID NO: 94, CDR2is represented by SEQ ID NO: 118, and CDR3 is represented by SEQ ID NO:134.

In a further aspect, the present invention provides a polypeptide asdescribed herein, which is a Nanobody, a V_(HH), a humanized V_(HH), ora camelized V_(H).

In a further aspect, the present invention provides a polypeptide asdescribed herein, further comprising a serum protein binding moiety.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said serum protein binding moiety binds serumalbumin.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said serum protein binding moiety is an ISVthat binds serum albumin.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said ISV that binds serum albumin essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich CDR1 is SFGMS (SEQ ID NO: 373), CDR2 is SISGSGSDTLYADSVKG (SEQ IDNO: 374) and CDR3 is GGSLSR (SEQ ID NO: 375), CDR as determinedaccording to Kabat definition; and/or in which CDR1 is GFTFRSFGMS (SEQID NO: 376) or GFTFRSFGMS (SEQ ID NO: 377), CDR2 is SISGSGSDTL (SEQ IDNO: 378) and CDR3 is GGSLSR (SEQ ID NO: 375), CDR as determinedaccording to Kontermann 2010.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said ISV that binds serum albumin is selectedfrom Alb8, Alb23, Alb129, Alb132, Alb11, Alb11 (S112K)-A, Alb82,Alb82-A, Alb82-AA, Alb82-AAA, Atb82-G, Alb82-GG, and Alb82-GGG (SEQ IDNOs: 348 to 360).

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said ISV is directly linked or is linked via alinker.

In a further aspect, the present invention provides a polypeptide asdescribed herein, wherein said linker is chosen from the groupconsisting of linkers of 5GS, 7GS, 9GS, 10GS, 15GS, 1865, 20GS, 25G5,30GS and 35GS (SEQ ID NOs: 362 to 372).

In a further aspect, the present invention provides a polypeptide asdescribed herein, further comprising a PEG moiety.

In a further aspect, the present invention provides a nucleic acid ornucleic acid sequence encoding a polypeptide as defined herein.

In a further aspect, the present invention provides vector comprising anucleic acid or nucleic acid sequence as defined herein.

In a further aspect, the present invention provides a host celltransformed or transfected with the nucleic acid or nucleic acidsequence as defined herein or with the vector as defined herein.

In a further aspect, the present invention provides a process for theproduction of the polypeptide as defined herein, said process comprisingculturing a host cell as defined herein under conditions allowing theexpression of the polypeptide as defined herein and recovering theproduced polypeptide from the culture.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising the polypeptide as described herein, or thepolypeptide produced according to the process as described herein.

In a further aspect, the present invention provides a polypeptide asdescribed herein, or produced as described herein, for use in treating asubject in need thereof.

In a further aspect, the present invention provides a method fordelivering a prophylactic or therapeutic polypeptide to a specificlocation, tissue or cell type in the body, the method comprising thesteps of administering to a subject a polypeptide as described herein,or produced as described herein.

In a further aspect, the present invention provides a polypeptide asdescribed herein, or produced as described herein for use in theprevention, treatment or amelioration of a disease selected from thegroup consisting of a proliferative disease, an inflammatory disease, aninfectious disease and an autoimmune disease.

In a further aspect, the present invention provides a method for theprevention, treatment or amelioration of a disease selected from thegroup consisting of a proliferative disease, an inflammatory disease, aninfectious disease and an autoimmune disease, comprising the step ofadministering to a subject in need thereof the polypeptide as describedherein, or produced as described herein.

In a further aspect, the present invention provides a polypeptide foruse in or a method for the prevention, treatment or amelioration of adisease as described herein, wherein said proliferative disease iscancer.

In a further aspect, the present invention provides a polypeptide foruse in or a method for the prevention, treatment or amelioration of adisease as described herein, wherein said cancer is chosen from thegroup consisting of carcinomas, gliomas, mesotheliomas, melanomas,lymphomas, leukemias, adenocarcinomas: breast cancer, ovarian cancer,cervical cancer, glioblastoma, multiple myeloma (including monoclonalgammopathy of undetermined significance, asymptomatic and symptomaticmyeloma), prostate cancer, and Burkitt's lymphoma, head and neck cancer,colon cancer, colorectal cancer, non-small cell lung cancer, small celllung cancer, cancer of the esophagus, stomach cancer, pancreatic cancer,hepatobiliary cancer, cancer of the gallbladder, cancer of the smallintestine, rectal cancer, kidney cancer, bladder cancer, prostatecancer, penile cancer, urethral cancer, testicular cancer, vaginalcancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenalcancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skincancer, retinoblastomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma,Kaposi's sarcoma, multicentric Castleman's disease or AIDS-associatedprimary effusion lymphoma, neuroectodermal tumors, rhabdomyosarcoma; aswell as any metastasis of any of the above cancers, as well asnon-cancer indications such as nasal polyposis.

In a further aspect, the present invention provides a polypeptide foruse in or a method for the prevention, treatment or amelioration of adisease as described herein, wherein the treatment is a combinationtreatment.

In a further aspect, the present invention provides a kit comprising apolypeptide as defined herein, a nucleic acid or nucleic acid sequenceas defined herein, a vector as defined herein, or a host cell as definedherein.

FIGURE LEGENDS

FIG. 1: QC of human TCR/CD3 and human CD3 transfected cell lines using100 nM of anti-human TCR α/β antibody (clone BW242/412) (black) and 100nM anti-human CD3 antibody (clone OKT-3) (grey). The MCF value (meanchannel fluorescence) is plotted for each cell line.

FIGS. 2A and 2B: Dose dependent binding of monovalent CD3 Nanobodies tohuman TCR/CD3 expressed on CMO-K1 cells (FIG. 2A) and to purifiedprimary human T cells (FIG. 28). The MCF value (mean channelfluorescence) is plotted against the concentration of the Nanobody.

FIG. 3: Dose dependent binding of monovalent CD3 Nanobodies to HEK293Hhuman TCR(2IAL)/CD3 (closed circle), HEK293H human CD3 (cross) and toHEK293H parental cell line (open circles). The MCF value (mean channelfluorescence) is plotted against the concentration of the Nanobody.

FIGS. 4A and 4B: T cell activation data of bead coupled monovalent CD3Nanobodies (FIG. 4A). T cell activation data of monovalent CD3Nanobodies presented in solution (FIG. 4B). Activation is measured bymonitoring the CD69 upregulation on primary human T cells. The MCF value(mean channel fluorescence) is plotted for each Nanobody.

FIGS. 5A-5C: Binding of a dilution series of CD20×CD3 (full line) andCD3×CD20 (dotted line) bispecific Nanobodies to human TCR/CD3 expressedon CHO-K1 cells (FIG. 5A), primary human T cells (FIG. 5B) and Ramoscells (FIG. 5C). The MCF value (mean channel fluorescence) is plottedagainst the concentration of the Nanobody.

FIGS. 6A and 6B: Dose-dependent killing effect of CD20×CD3 (full line)and CD3×CD20 (dotted line) bispecific Nanobodies in a flow cytometrybased human T cells mediated Ramos (FIG. 6A) and Raji (FIG. 68) B cellkilling assay. The % cell death (% of TOPRO positive cells) is plottedagainst the concentration of the Nanobody.

FIG. 7: Dose-dependent binding of the anti-CD20 Nanobody on human CD20Ramos (open symbols) and Raji (closed symbols) cells. The MCF value(mean channel fluorescence) is plotted against the concentration of theNanobody.

FIG. 8: Dose-dependent killing effect of CD20×CD3 (full line) andCD3×CD20 (dotted line) bispecific Nanobodies in the xCELLigence basedhuman T cells mediated CHO-K1 human CD20 killing assay assay. The CI isplotted against the concentration of Nanobody.

FIG. 9: Killing effect of 1 μM of CD20×CD3 and an irrelevant constructin a xCELLigence based killing assay using CHO-K1 human CD20 cells(black bars) and using CHO-K1 parental cell line (grey bars) toillustrate TAA dependent killing. The cell index (CI) is plotted againstthe concentration of the Nanobody.

FIG. 10: Dose-dependent killing effect of CD20×CD3 Nanobodies with 9GSlinker (open circles-dotted line) and a 35GS linker (closedsquares-dotted line) and the CD3×CD20 Nanobody with a 35GS linker(closed diamonds-solid line) in a flow cytometry based killing assayusing Ramos. The % cell death (TORPRO positive cells) is plotted againstthe concentration of Nanobody.

FIG. 11: Dose-dependent killing of 7017000062 in a flow cytometry basedhuman T cells mediated Ramos B cell killing assay using differenteffector (E) to target (T) ratio's (E:T ratio 10:1—closed circles, E:Tratio 5:1—open squares, E:T ratio 2:1—closed triangles and E:T ratio1:1—open diamonds. The % cell death (% of TOPRO positive cells) isplotted against the concentration of the Nanobody.

FIG. 12: Time-dependent cytolytic activity of CD20/CD3 in the purifiedprimary human T cells mediated killing assay in xCELLigence using CHO-K1human CD20 target cells. The % specific lysis is plotted against theconcentration of the construct. The different curves represent theanalysis time after addition of the T cells.

FIGS. 13A-13C: Binding of a serial dilution of HLE constructs to humanTCR/CD3 expressed on CHO-K1 cells (FIG. 13A), primary human T cells(FIG. 13B) and Ramos cells (FIG. 13C). The MCF value (mean channelfluorescence) is plotted against the concentration of the Nanobody.

FIGS. 14A-14D: Dose-dependent killing of CD20×CD3 bispecific Nanobody(solid line-diamonds) versus CD20×CD3×ALB11 constructs (solidline-closed triangle) (FIG. 14A, FIG. 14C) and dose-dependent killingeffect of CD20×CD3×ALB11 constructs in the absence (solid line-closedtriangle) or presence of 30 μM HSA (dotted line-open triangle) in a flowcytometry based human T cells mediated Ramos B cell killing assay (FIG.14B, FIG. 14D). The % cell death (% of TOPRO positive cells) is plottedagainst the concentration of the Nanobody.

FIG. 15: Binding of 100 nM monovalent anti-HER2 Nanobody (SF07) toSK-BR-3, MCF-7 and MDA-MB-468 cell lines in flow cytometry to compareHER2 expression levels. The MCF value (mean channel fluorescence) isplotted for each cell line.

FIG. 16: Dose-dependent killing effect of bispecific CD3×HER2 Nanobodies(dotted line) and bispecific HER2×CD3 (full line) in an xCELLigencebased human T cells mediated cell killing assay. Data were analysedusing at 18 h. The Cell index (CI) was plotted against the concentrationof the Nanobody.

FIG. 17: Dose-dependent INF-γ production by human T cells afterincubation of human CD20 positive CHO-K1 cells with bispecific CD20×CD3Nanobodies in flow cytometry based killing assay. Data were analysedafter 72 h incubation. The OD at 405 nm was plotted against theconcentration of the Nanobody.

FIG. 18: Study design PBMC B cell depletion model. PBMCs were injectedintraperitoneally to animals on day 3 (D3). Mice were treated from D3 toD7 with T017000084 (CD3/CD20) IV Q1Dx5 or T017000088 IV Q1Dx5(irrelevant Nanobody).

FIG. 19: Absolute PBMC-derived B cell count on log scale. Individualanimal results are depicted. The number of B-cells is shown in functionof the different treatment groups.

FIG. 20: Study design Ramos model. Ramos cells were injectedintravenously to mice on Dl. PBMCs were injected intraperitoneally toanimals on D3. Mice were treated from D3 to D7 with T0174000084(CD3/CD20) IV Q1Dx5 or T017000088 IV Q1Dx5 (irrelevant Nanobody).

FIG. 21: Absolute Ramos B cell count on log scale. Individual animalresults are depicted. The open circles on top of the graph show whichactive doses were statistically significant different from theirrelevant NB (T017000088) based on the F-tests from the mixed-effectsANOVA analysis. All effects are statistically significant at the 5%level of significance.

FIG. 22: Absolute PBMC-derived B cell count on log scale. Individualanimal results are depicted. The open circles on top of the graph showwhich active doses were statistically significant different from theirrelevant NB (T017000088) based on the F-tests from the mixed-effectsANOVA analysis. All effects are statistically significant at the 5%level of significance.

FIGS. 23A and 23B: Determination of EGFR (FIG. 23A; Santa Cruz, sc-120PE) or CEACAM5 (FIG. 23B; Sino Biological, 11077-MM02-P) expressionlevel on HER14, Hela, LoVo and L5174-T cell lines in flow cytometry. TheMCF value (mean channel fluorescence) is plotted for each cell line.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors realized that formats bringing T cells and tumourcells together to induce an immune response should comply with variousand frequently opposing requirements. The format should be broadlyapplicable. In particular, the format should preferably be useful in abroad range of patients and preferably also against a broad range oftumours. The format should preferably be safe and only target theintended cells. In addition, the format should preferably be smallenough to easily penetrate tissues and tumours, while on the other handthe format should be patient friendly. For instance, the format shouldhave an extended half-life, such that the format is not removedinstantaneously upon administration by renal clearance. However,extending the half-life should preferably not introduce off-targetactivity and side effects or limit the penetration into tissues andtumours. Additionally, it was recognized that tumour cells often createescape mechanisms by the down-regulation of targeted antigens within atherapy. Accordingly, in a further preferred version, the format shouldtarget simultaneously multiple antigens.

The present invention realizes at least one of these requirements.

In particular, it was hypothesized that immunoglobulin single variabledomains (ISVs) would in principle be ideal candidates, since they aresmall enough to easily penetrate (tumour) tissue and can be combinedwith other 15Vs as building blocks. Next, ISVs directed against CD3, andin particular CD3E, should have broad applicability.

Six clusters of related ISVs were identified, which had an unexpectedrange of advantageous features. First, the ISVs were unexpectedlybroadly applicable, i.e. the CD3 ISVs were able to bind to T cells fromdifferent donors with high affinity. Formatted in a multispecificpolypeptide, the CD3 ISVs enabled tumour cell killing with differenttumour associated antigens. Hence, the CD3 ISVs can be used against amultitude of cancers. In addition, the multispecific polypeptidescomprising the CD3 ISVs remained active when bound to albumin. Thiscontributes to a favourable PK profile and patient compliance, whileminimizing side effects. The polypeptides of the invention only showedeffects when bound both to the T cell and the target cell, which isindicative of its safety.

The present inventors considered that the simultaneous targeting ofmultiple antigens reduces the probability of generating tumour escapevariants, because of which the therapeutic activity of T cell engagingstrategy is improved. Multispecific polypeptides are provided whichcomprise a CD3 ISV combined with immunoglobulin single variable domainsagainst different target antigens and/or different epitopes on aparticular antigen (biparatopic).

Immunoglobulin sequences, such as antibodies and antigen bindingfragments derived there from (e.g., immunoglobulin single variabledomains or ISVs) are used to specifically target their respectiveantigens in research and therapeutic applications. The generation ofimmunoglobulin single variable domains such as e.g., VHHs or Nanobodiesmay involve the immunization of an experimental animal such as a Llama,construction of phage libraries from immune tissue, selection of phagedisplaying antigen binding immunoglobulin single variable domains andscreening of said domains and engineered constructs thereof for thedesired specificities (WO 94/04678). Alternatively, similarimmunoglobulin single variable domains such as e.g., dAbs can begenerated by selecting phage displaying antigen binding immunoglobulinsingle variable domains directly from naive or synthetic libraries andsubsequent screening of said domains and engineered constructs thereoffor the desired specificities (Ward et al., Nature, 1989, 341: 544-6;Holt et al., Trends Biotechnol., 2003, 21(11):484-490; as well as forexample WO 06/030220, WO 06/003388 and other published patentapplications of Domantis Ltd.). Unfortunately, the use of monoclonaland/or heavily engineered antibodies also carries a high manufacturingcost and may result in suboptimal tumor penetration compared to otherstrategies.

The present invention provides multispecific polypeptides thatspecifically bind to CD3 of the T cell receptor complex, with anunexpected range of advantageous features. First, the polypeptides areeasy to manufacture. Moreover, the ISVs are unexpectedly broadapplicable, i.e. the CD3 ISVs were able to bind to T cells fromdifferent donors with high affinity. Formatted in a multispecificpolypeptide, the CD3 ISVs enabled tumour cell killing with differenttumour associated antigens. In contrast, no killing was observed whenthe polypeptides were not bound to T cells and target cell whichunderscores the safety of the polypeptides of the invention. Hence, theCD3 ISVs can be used against a multitude of cancers. Moreover, the CD3ISVs can be considered as safe. In addition, the multispecificpolypeptides comprising the CD3 ISVs remained active when bound toalbumin. This will contribute to a favourable PK profile and patientcompliance, while minimizing side effects. Accordingly, the presentinvention relates to a polypeptide comprising a first and a secondimmunoglobulin single variable domain (ISV), wherein the first ISV hashigh affinity for/binds to CD3 and the second ISV has high affinityfor/binds to an antigen on a cell (target cell), preferably a tumourcell. The antigen is preferably specific for said target cell, such ase.g. a tumour associated antigen (TAA). The multispecific polypeptide ofthe invention directs the T cell to the cell, e.g. a tumour cell andinduces T cell activation in order to allow said T cell to inhibit orkill said target cell, e.g. said tumour cell.

Definitions

-   a) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks mentioned in    paragraph a) on page 46 of WO 08/020079.-   b) The term “immunoglobulin single variable domain”, interchangeably    used with “single variable domain” and “ISV”, defines molecules    wherein the antigen binding site is present on, and formed by, a    single immunoglobulin domain. This sets immunoglobulin single    variable domains apart from “conventional” immunoglobulins or their    fragments (such as Fabs, scFvs, etc.), wherein two immunoglobulin    domains, in particular two variable domains, interact to form an    antigen binding site. Typically, in conventional immunoglobulins, a    heavy chain variable domain (VH) and a light chain variable domain    (VL) interact to form an antigen binding site. In this case, the    complementarity determining regions (CDRs) of both VH and VL will    contribute to the antigen binding site, i.e. a total of 6 CDRs will    be involved in antigen binding site formation. In contrast, the    binding site of an immunoglobulin single variable domain is formed    by a single VH or VL domain. Hence, the antigen binding site of an    immunoglobulin single variable domain is formed by no more than    three CDRs.    -   The terms “immunoglobulin single variable domain”, “single        variable domain”, and “ISV” hence do not comprise conventional        immunoglobulins or their fragments which require interaction of        at least two variable domains for the formation of an antigen        binding site. However, these terms do comprise fragments of        conventional immunoglobulins wherein the antigen binding site is        formed by a single variable domain.    -   The term “immunoglobulin single variable domain” or “ISV”        includes (without being limiting) antigen-binding domains or        fragments such as V_(HH) domains or V_(H) or V_(L) domains,        respectively. The terms antigen-binding molecules or        antigen-binding protein are used interchangeably and include        also the term Nanobodies. The immunoglobulin single variable        domains can be light chain variable domain sequences (e.g., a        V_(L)-sequence), or heavy chain variable domain sequences (e.g.,        a V_(H)-sequence); more specifically, they can be heavy chain        variable domain sequences that are derived from a conventional        four-chain antibody or heavy chain variable domain sequences        that are derived from a heavy chain antibody. Accordingly, the        immunoglobulin single variable domains can be domain antibodies,        or immunoglobulin sequences that are suitable for use as domain        antibodies, single domain antibodies, or immunoglobulin        sequences that are suitable for use as single domain antibodies,        “dAbs”, or immunoglobulin sequences that are suitable for use as        dAbs, or Nanobodies, including but not limited to V_(HH)        sequences, humanized VHH sequences or camelized VH sequences.        The invention includes immunoglobulin sequences of different        origin, comprising mouse, rat, rabbit, donkey, human and camelid        immunoglobulin sequences. The immunoglobulin single variable        domain includes fully human, humanized, otherwise sequence        optimized or chimeric immunoglobulin sequences. The        immunoglobulin single variable domain and structure of an        immunoglobulin single variable domain can be considered—without        however being limited thereto—to be comprised of four framework        regions or “FR's”, which are referred to in the art and herein        as “Framework region 1” or “FR1”; as “Framework region 2” or        “FR2”; as “Framework region 3” or “FR3”; and as “Framework        region 4” or “FR4”, respectively; which framework regions are        interrupted by three complementary determining regions or        “CDR's”, which are referred to in the art as “Complementarity        Determining Region 1” or “CDR1”; as “Complementarity Determining        Region 2” or “CDR2”; and as “Complementarity Determining Region        3” or “CDR3”, respectively. It is noted that the terms Nanobody        or Nanobodies are registered trademarks of Ablynx N.V. and thus        may also be referred to as Nanobody® or Nanobodies®,        respectively.-   c) Unless indicated otherwise, the terms “immunoglobulin sequence”,    “sequence”, “nucleotide sequence” and “nucleic acid” are as    described in paragraph b) on page 46 of WO 08/020079.-   d) Unless indicated otherwise, all methods, steps, techniques and    manipulations that are not specifically described in detail can be    performed and have been performed in a manner known per se, as will    be clear to the skilled person. Reference is for example again made    to the standard handbooks and the general background art mentioned    herein and to the further references cited therein; as well as to    for example the following reviews Presta 2006 (Adv. Drug Deliv. Rev.    58 (5-6):640-656), Levin and Weiss 2006 (Mol. Biosyst. 2(1):49-57),    Irving et al. 2005 (J. Immunol. Methods 248(1-2):31-45), Schmitz et    al. 2000 (Placenta 21 Suppl. A: 5106-112, Gonzales et al. 2005    (Tumour Biol. 26(1):31-43), which describe techniques for protein    engineering, such as affinity maturation and other techniques for    improving the specificity and other desired properties of proteins    such as immunoglobulins.-   e) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code. Reference is made to    Table A-2 on page 48 of the International application WO 08/020079    of Ablynx N.V. entitled “immunoglobulin single variable domains    directed against IL-6R and polypeptides comprising the same for the    treatment of diseases and disorders associated with 11-6 mediated    signalling”.-   f) For the purposes of comparing two or more nucleotide sequences,    the percentage of “sequence identity” between a first nucleotide    sequence and a second nucleotide sequence may be calculated or    determined as described in paragraph e) on page 49 of WO 08/020079    (incorporated herein by reference), such as by dividing [the number    of nucleotides in the first nucleotide sequence that are identical    to the nucleotides at the corresponding positions in the second    nucleotide sequence] by [the total number of nucleotides in the    first nucleotide sequence] and multiplying by [100%], in which each    deletion, insertion, substitution or addition of a nucleotide in the    second nucleotide sequence—compared to the first nucleotide    sequence—is considered as a difference at a single nucleotide    (position); or using a suitable computer algorithm or technique,    again as described in paragraph e) on pages 49 of WO 08/020079    (incorporated herein by reference).-   g) For the purposes of comparing two or more immunoglobulin single    variable domains or other amino acid sequences such e.g. the    polypeptides of the invention etc., the percentage of “sequence    identity” between a first amino acid sequence and a second amino    acid sequence (also referred to herein as “amino acid identity”) may    be calculated or determined as described in paragraph f) on pages 49    and 50 of WO 08/020079 (incorporated herein by reference), such as    by dividing [the number of amino acid residues in the first amino    acid sequence that are identical to the amino acid residues at the    corresponding positions in the second amino acid sequence] by [the    total number of amino acid residues in the first amino acid    sequence] and multiplying by [100%], in which each deletion,    insertion, substitution or addition of an amino acid residue in the    second amino acid sequence—compared to the first amino acid    sequence—is considered as a difference at a single amino acid    residue (position), i.e., as an “amino acid difference” as defined    herein; or using a suitable computer algorithm or technique, again    as described in paragraph f) on pages 49 and 50 of WO 08/020079    (incorporated herein by reference).    -   Also, in determining the degree of sequence identity between two        immunoglobulin single variable domains, the skilled person may        take into account so-called “conservative” amino acid        substitutions, as described on page 50 of WO 08/020079.    -   Any amino acid substitutions applied to the polypeptides        described herein may also be based on the analysis of the        frequencies of amino acid variations between homologous proteins        of different species developed by Schulz et al. 1978 (Principles        of Protein Structure, Springer-Verlag), on the analyses of        structure forming potentials developed by Chou and Fasman 1975        (Biochemistry 13: 211) and 1978 (Adv. Enzymol. 47: 45-149), and        on the analysis of hydrophobicity patterns in proteins developed        by Eisenberg et al. 1984 (Proc. Natl. Acad. Sci. USA 81:        140-144), Kyte & Doolittle 1981 (J Molec. Biol. 157: 105-132),        and Goldman et al. 1986 (Ann. Rev. Biophys. Chem. 15: 321-353),        all incorporated herein in their entirety by reference.        Information on the primary, secondary and tertiary structure of        Nanobodies is given in the description herein and in the general        background art cited above. Also, for this purpose, the crystal        structure of a V_(HH) domain from a llama is for example given        by Desmyter et al. 1996 (Nature Structural Biology, 3: 803),        Spinelli et al. 1996 (Natural Structural Biology 3: 752-757),        and Decanniere et al. 1999 (Structure, 7: 361). Further        information about some of the amino acid residues that in        conventional V_(H) domains form the V_(H)/V_(L) interface and        potential camelizing substitutions on these positions can be        found in the prior art cited above.-   h) Immunoglobulin single variable domains and nucleic acid sequences    are said to be “exactly the same” if they have 100% sequence    identity (as defined herein) over their entire length.-   i) When comparing two immunoglobulin single variable domains, the    term “amino acid difference” refers to an insertion, deletion or    substitution of a single amino acid residue on a position of the    first sequence, compared to the second sequence; it being understood    that two immunoglobulin single variable domains can contain one, two    or more such amino acid differences.-   j) When a nucleotide sequence or amino acid sequence is said to    “comprise” another nucleotide sequence or amino acid sequence,    respectively, or to “essentially consist of” another nucleotide    sequence or amino acid sequence, this has the meaning given in    paragraph i) on pages 51-52 of WO 08/020079.-   k) The term “in essentially isolated form” has the meaning given to    it in paragraph j) on pages 52 and 53 of WO 08/020079.-   l) The terms “domain” and “binding domain” have the meanings given    to it in paragraph k) on page 53 of WO 08/020079.-   m) The terms “antigenic determinant” and “epitope”, which may also    be used interchangeably herein, have the meanings given to it in    paragraph l) on page 53 of WO 08/020079.-   n) As further described in paragraph m) on page 53 of WO 08/020079,    an amino acid sequence (such as an antibody, a polypeptide of the    invention, or generally an antigen-binding protein or polypeptide or    a fragment thereof) that can (specifically) bind to, that has    affinity for and/or that has specificity for a specific antigenic    determinant, epitope, antigen or protein (or for at least one part,    fragment or epitope thereof) is said to be “against” or “directed    against” said antigenic determinant, epitope, antigen or protein.-   o) The term “specificity” refers to the number of different types of    antigens or antigenic determinants to which a particular    antigen-binding molecule or antigen-binding protein (such as an ISV,    Nanobody or a polypeptide of the invention) can bind. The    specificity of an antigen-binding protein can be determined based on    affinity and/or avidity.    -   The affinity, represented by the equilibrium constant for the        dissociation of an antigen with an antigen-binding protein        (K_(D) or KD), is a measure for the binding strength between an        antigenic determinant, i.e. the target, and an antigen-binding        site on the antigen-binding protein, i.e. the ISV or Nanobody:        the lesser the value of the K_(D), the stronger the binding        strength between an antigenic determinant and the        antigen-binding molecule (alternatively, the affinity can also        be expressed as the affinity constant (K_(A)), which is        1/K_(D)). As will be clear to the skilled person (for example on        the basis of the further disclosure herein), affinity can be        determined in a manner known per se, depending on the specific        antigen of interest.    -   Avidity is the affinity of the polypeptide, i.e. the ligand is        able to bind via two (or more) pharmacophores (ISV) in which the        multiple interactions synergize to enhance the “apparent”        affinity. Avidity is the measure of the strength of binding        between the polypeptide of the invention and the pertinent        antigens. The polypeptide of the invention is able to bind via        its two (or more) building blocks, such as ISVs or Nanobodies,        to the at least two targets, in which the multiple interactions,        e.g. the first building block, ISV or Nanobody binding to the        first target and the second building block, ISV, or Nanobody        binding to the second target, synergize to enhance the        “apparent” affinity. Avidity is related to both the affinity        between an antigenic determinant and its antigen binding site on        the antigen-binding molecule and the number of pertinent binding        sites present on the antigen-binding molecules. For example, and        without limitation, polypeptides that contain two or more        building blocks, such as ISVs or Nanobodies directed against        different targets on a cell may (and usually will) bind with        higher avidity than each of the individual monomers or        individual building blocks, such as, for instance, the        monovalent ISVs or Nanobodies, comprised in the polypeptides of        the invention.    -   Any K_(D) value greater than 10⁻⁴ mol/liter (or any K_(A) value        lower than 10⁴ M⁻¹) liters/mol is generally considered to        indicate non-specific binding.    -   The polypeptides of the invention comprise a first and a second        building block, e.g. a first and a second ISV, or a first and a        second Nanobody. Preferably the affinity of each building block,        e.g. ISV or Nanobody, is determined individually. In other        words, the affinity is determined for the monovalent building        block, ISV or Nanobody, independent of avidity effects due to        the other building block, ISV or Nanobody, which might or might        not be present. The affinity for a monovalent building block,        ISV or Nanobody can be determined on the monovalent building        block, ISV or Nanobody per se, i.e. when said monovalent        building block, ISV or Nanobody is not comprised in the        polypeptide of the invention. In the alternative or in addition,        the affinity for a monovalent building block, ISV or Nanobody        can be determined on one target while the other target is        absent.    -   The binding of an antigen-binding protein to an antigen or        antigenic determinant can be determined in any suitable manner        known per se, including, for example, Scatchard analysis and/or        competitive binding assays, such as radioimmunoassays (RIA),        enzyme immunoassays (EIA) and sandwich competition assays, and        the different variants thereof known per se in the art; as well        as the other techniques mentioned herein.    -   The dissociation constant may be the actual or apparent        dissociation constant, as will be clear to the skilled person.        Methods for determining the dissociation constant will be clear        to the skilled person, and for example include the techniques        mentioned herein. In this respect, it will also be clear that it        may not be possible to measure dissociation constants of more        than 10⁻⁴ moles/liter or 10⁻³ moles/liter (e.g., of 10⁻²        moles/liter). Optionally, as will also be clear to the skilled        person, the (actual or apparent) dissociation constant may be        calculated on the basis of the (actual or apparent) association        constant (K_(A)), by means of the relationship [K_(D)=1/K_(A)].    -   The affinity denotes the strength or stability of a molecular        interaction. The affinity is commonly given as by the K_(D), or        dissociation constant, which has units of mol/liter (or M). The        affinity can also be expressed as an association constant,        K_(A), which equals 1/K_(D) and has units of (mol/liter)⁻¹ (or        M⁻¹). In the present specification, the stability of the        interaction between two molecules (such as an amino acid        sequence, Nanobody or polypeptide of the invention and its        intended target) will mainly be expressed in terms of the K_(D)        value of their interaction; it being clear to the skilled person        that in view of the relation K_(A)=1/K_(D), specifying the        strength of molecular interaction by its K_(D) value can also be        used to calculate the corresponding K_(A) value. The K_(D)-value        characterizes the strength of a molecular interaction also in a        thermodynamic sense as it is related to the free energy (DG) of        binding by the well-known relation DG=RT·ln(K_(D)) (equivalently        DG=−RT·ln(K_(A))), where R equals the gas constant, T equals the        absolute temperature and ln denotes the natural logarithm.    -   The K_(D) for biological interactions which are considered        meaningful (e.g. specific) are typically in the range of 10⁻¹⁰M        (0.1 nM) to 10⁻⁵M (10000 nM). The stronger an interaction is,        the lower is its K_(D).    -   The K_(D) can also be expressed as the ratio of the dissociation        rate constant of a complex, denoted as k_(off), to the rate of        its association, denoted k_(on) (so that K_(D)=k_(off)/k_(on)        and K_(A)=K_(on)/k_(off)). The off-rate k_(off) has units s⁻¹        (where s is the SI unit notation of second). The on-rate k_(on)        has units M⁻¹s⁻¹. The on-rate may vary between 10² M⁻¹s⁻¹ to        about 10⁷ M⁻³s⁻¹, approaching the diffusion-limited association        rate constant for biomolecular interactions. The off-rate is        related to the half-life of a given molecular interaction by the        relation t_(1/2)=ln(2)/k_(off). The off-rate may vary between        10⁻⁶s⁻¹ (near irreversible complex with a t_(1/2) of multiple        days) to 1s⁻¹ (t_(1/2)=0.69s).    -   The affinity of a molecular interaction between two molecules        can be measured via different techniques known per se, such as        the well-known surface plasmon resonance (SPR) biosensor        technique (see for example Ober et al. 2001, Intern. Immunology,        13: 1551-1559). The term “surface plasmon resonance”, as used        herein, refers to an optical phenomenon that allows for the        analysis of real-time biospecific interactions by detection of        alterations in protein concentrations within a biosensor matrix,        where one molecule is immobilized on the biosensor chip and the        other molecule is passed over the immobilized molecule under        flow conditions yielding k_(on), k_(off) measurements and hence        K_(D) (or K_(A)) values. This can for example be performed using        the well-known BIAcore® system (BIAcore International AB, a GE        Healthcare company, Uppsala, Sweden and Piscataway, N.J.). For        further descriptions, see Jonsson et al. 1993 (Ann. Biol. Clin.        51: 19-26), Jonsson et al. 1991 (Biotechniques 11: 620-627),        Johnnson, et al. 1995 (J. Mol. Recognit. 8: 125-131), and        Johnnson, et al. 1991 (Anal. Biochem. 198: 268-277).    -   Alternatively, affinities can be measured in Kinetic Exclusion        Assay (KinExA) (see for example Drake et al. 2004, Anal.        Biochem., 328: 35-43), using the KinExA® platform (Sapidyne        Instruments Inc, Boise, USA). The term “KinExA”, as used herein,        refers to a solution-based method to measure true equilibrium        binding affinity and kinetics of unmodified molecules.        Equilibrated solutions of an antibody/antigen complex are passed        over a column with beads precoated with antigen (or antibody),        allowing the free antibody (or antigen) to bind to the coated        molecule. Detection of the antibody (or antigen) thus captured        is accomplished with a fluorescently labeled protein binding the        antibody (or antigen).    -   It will also be clear to the skilled person that the measured        K_(D) may correspond to the apparent K_(D) if the measuring        process somehow influences the intrinsic binding affinity of the        implied molecules for example by artefacts related to the        coating on the biosensor of one molecule.

Also, an apparent K_(D) may be measured if one molecule contains morethan one recognition site for the other molecule. In such situation themeasured affinity may be affected by the avidity of the interaction bythe two molecules.

-   -   Another approach that may be used to assess affinity is the        2-step ELISA (Enzyme-Linked Immunosorbent Assay) procedure of        Friguet et al. 1985 (J. Immunol. Methods, 77: 305-19). This        method establishes a solution phase binding equilibrium        measurement and avoids possible artefacts relating to adsorption        of one of the molecules on a support such as plastic.    -   However, the accurate measurement of K_(D) may be quite        labour-intensive and, as consequence, often apparent K_(D)        values are determined to assess the binding strength of two        molecules. It should be noted that as long all measurements are        made in a consistent way (e.g. keeping the assay conditions        unchanged) apparent K_(D) measurements can be used as an        approximation of the true K_(D) and hence, in the present        document, K_(D) and apparent K_(D) should be treated with equal        importance or relevance.    -   Finally, it should be noted that in many situations the        experienced scientist may judge it to be convenient to determine        the binding affinity relative to some reference molecule. For        example, to assess the binding strength between molecules A and        B, one may e.g. use a reference molecule C that is known to bind        to B and that is suitably labelled with a fluorophore or        chromophore group or other chemical moiety, such as biotin for        easy detection in an ELISA or FACS (Fluorescent activated cell        sorting) or other format (the fluorophore for fluorescence        detection, the chromophore for light absorption detection, the        biotin for streptavidin-mediated ELISA detection). Typically,        the reference molecule C is kept at a fixed concentration and        the concentration of A is varied for a given concentration or        amount of B. As a result an IC₅₀ value is obtained corresponding        to the concentration of A at which the signal measured for C in        absence of A is halved. Provided K_(D ref), the K_(D) of the        reference molecule, is known, as well as the total concentration        c_(ref) of the reference molecule, the apparent K_(D) for the        interaction A-B can be obtained from following formula:        K_(D)=IC₅₀/(1+c_(ref)/K_(D ref)). Note that if        c_(ref)<<K_(D ref), K_(D)≈IC₅₀. Provided the measurement of the        IC₅₀ is performed in a consistent way (e.g. keeping c_(ref)        fixed) for the binders that are compared, the strength or        stability of a molecular interaction can be assessed by the IC₅₀        and this measurement is judged as equivalent to K_(D) or to        apparent K_(D) throughout this text.

-   p) The half-life of an amino acid sequence, compound or polypeptide    of the invention can generally be defined as described in    paragraph o) on page 57 of WO 08/020079 and as mentioned therein    refers to the time taken for the serum concentration of the amino    acid sequence, compound or polypeptide to be reduced by 50%, in    vivo, for example due to degradation of the sequence or compound    and/or clearance or sequestration of the sequence or compound by    natural mechanisms. The in vivo half-life of an amino acid sequence,    compound or polypeptide of the invention can be determined in any    manner known per se, such as by pharmacokinetic analysis. Suitable    techniques will be clear to the person skilled in the art, and may    for example generally be as described in paragraph o) on page 57 of    WO 08/020079. As also mentioned in paragraph o) on page 57 of WO    08/020079, the half-life can be expressed using parameters such as    the t½-alpha, t½-beta and the area under the curve (AUC). Reference    is for example made to the Experimental Part below, as well as to    the standard handbooks, such as Kenneth et al. 1996 (Chemical    Stability of Pharmaceuticals: A Handbook for Pharmacists) and Peters    et al. 1996 (Pharmacokinete analysis: A Practical Approach).    Reference is also made to Gibaldi & Perron 1982 (Pharmacokinetics,    Dekker M, 2nd Rev. edition). The terms “increase in half-life” or    “increased half-life” are as defined in paragraph o) on page 57 of    WO 08/020079 and in particular refer to an increase in the t½-beta,    either with or without an increase in the t½-alpha and/or the AUC or    both.

-   q) In respect of a target or antigen, the term “interaction site” on    the target or antigen means a site, epitope, antigenic determinant,    part, domain or stretch of amino acid residues on the target or    antigen that is a site for binding to a ligand, receptor or other    binding partner, a catalytic site, a cleavage site, a site for    allosteric interaction, a site involved in multimerisation (such as    homomerization or heterodimerization) of the target or antigen; or    any other site, epitope, antigenic determinant, part, domain or    stretch of amino acid residues on the target or antigen that is    involved in a biological action or mechanism of the target or    antigen. More generally, an “interaction site” can be any site,    epitope, antigenic determinant, part, domain or stretch of amino    acid residues on the target or antigen to which an amino acid    sequence or polypeptide of the invention can bind such that the    target or antigen (and/or any pathway, interaction, signalling,    biological mechanism or biological effect in which the target or    antigen is involved) is modulated.

-   r) An immunoglobulin single variable domain or polypeptide is said    to be “specific for” a first target or antigen compared to a second    target or antigen when it binds to the first antigen with an    affinity/avidity (as described above, and suitably expressed as a    K_(D) value, K_(A) value, K_(off) rate and/or K_(on) rate) that is    at least 10 times, such as at least 100 times, and preferably at    least 1000 times, and up to 10.000 times or more better than the    affinity with which said amino acid sequence or polypeptide binds to    the second target or polypeptide. For example, the first antigen may    bind to the target or antigen with a K_(D) value that is at least 10    times less, such as at least 100 times less, and preferably at least    1000 times less, such as 10.000 times less or even less than that,    than the K_(D) with which said amino acid sequence or polypeptide    binds to the second target or polypeptide. Preferably, when an    immunoglobulin single variable domain or polypeptide is “specific    for” a first target or antigen compared to a second target or    antigen, it is directed against (as defined herein) said first    target or antigen, but not directed against said second target or    antigen.

-   s) The terms “cross-block”, “cross-blocked” and “cross-blocking” are    used interchangeably herein to mean the ability of an immunoglobulin    single variable domain or polypeptide to interfere with the binding    of the natural ligand to its receptor(s). The extent to which an    immunoglobulin single variable domain or polypeptide of the    invention is able to interfere with the binding of another compound    such as the natural ligand to its target and therefore whether it    can be said to cross-block according to the invention, can be    determined using competition binding assays. One particularly    suitable quantitative cross-blocking assay uses a FACS- or an    ELISA-based approach or Alphascreen to measure competition between    the labelled (e.g., His tagged or biotinylated) immunoglobulin    single variable domain or polypeptide according to the invention and    the other binding agent in terms of their binding to the target. The    experimental part generally describes suitable FACS-, ELISA- or    Alphascreen-displacement-based assays for determining whether a    binding molecule cross-blocks or is capable of cross-blocking an    immunoglobulin single variable domain or polypeptide according to    the invention. It will be appreciated that the assay can be used    with any of the immunoglobulin single variable domains or other    binding agents described herein. Thus, in general, a cross-blocking    amino acid sequence or other binding agent according to the    invention is for example one which will bind to the target in the    above cross-blocking assay such that, during the assay and in the    presence of a second amino acid sequence or other binding agent of    the invention, the recorded displacement of the immunoglobulin    single variable domain or polypeptide according to the invention is    between 60% and 100% (e.g., in ELISA/Alphascreen based competition    assay) or between 80% to 100% (e.g., in FACS based competition    assay) of the maximum theoretical displacement (e.g. displacement by    cold (e.g., unlabeled) immunoglobulin single variable domain or    polypeptide that needs to be cross-blocked) by the to be tested    potentially cross-blocking agent that is present in an amount of    0.01 mM or less (cross-blocking agent may be another conventional    monoclonal antibody such as IgG, classic monovalent antibody    fragments (Fab, scFv) and engineered variants (e.g., diabodies,    triabodies, minibodies, VHHs, dAbs, VHs, Vis)).

-   t) An amino acid sequence such as e.g. an immunoglobulin single    variable domain or polypeptide according to the invention is said to    be a “VHH1 type immunoglobulin single variable domain” or “VHH type    1 sequence”, if said VHH1 type immunoglobulin single variable domain    or VHH type 1 sequence has 85% identity (using the VHH1 consensus    sequence as the query sequence and use the blast algorithm with    standard setting, i.e., blosom62 scoring matrix) to the VHH1    consensus sequence    (QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSD    GSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA) (SEQ ID NO: 361), and    mandatorily has a cysteine in position 50, i.e., C50 (using Kabat    numbering).

-   u) An amino acid sequence such as e.g., an immunoglobulin single    variable domain or polypeptide according to the invention is said to    be “cross-reactive” for two different antigens or antigenic    determinants (such as serum albumin from two different species of    mammal, such as human serum albumin and cynomolgus monkey serum    albumin) if it is specific for (as defined herein) both these    different antigens or antigenic determinants.

-   v) As further described in paragraph q) on pages 58 and 59 of WO    08/020079 (incorporated herein by reference), the amino acid    residues of an immunoglobulin single variable domain are numbered    according to the general numbering for V_(H) domains given by Kabat    et al. (“Sequence of proteins of immunological interest”, US Public    Health Services, NIH Bethesda, Md., Publication No. 91), as applied    to V_(HH) domains from Camelids in the article of Riechmann and    Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see for    example FIG. 2 of this publication). It should be noted that—as is    well known in the art for V_(H) domains and for VHH domains—the    total number of amino acid residues in each of the CDRs may vary and    may not correspond to the total number of amino acid residues    indicated by the Kabat numbering (that is, one or more positions    according to the Kabat numbering may not be occupied in the actual    sequence, or the actual sequence may contain more amino acid    residues than the number allowed for by the Kabat numbering). This    means that, generally, the numbering according to Kabat may or may    not correspond to the actual numbering of the amino acid residues in    the actual sequence. The total number of amino acid residues in a VH    domain and a VHH domain will usually be in the range of from 110 to    120, often between 112 and 115. It should however be noted that    smaller and longer sequences may also be suitable for the purposes    described herein.    -   Determination of CDR regions may also be done according to        different methods. In the CDR determination according to Kabat,        FR1 of an immunoglobulin single variable domain comprises the        amino acid residues at positions 1-30, CDR1 of an immunoglobulin        single variable domain comprises the amino acid residues at        positions 31-35, FR2 of an immunoglobulin single variable domain        comprises the amino acids at positions 36-49, CDR2 of an        immunoglobulin single variable domain comprises the amino acid        residues at positions 50-65, FR3 of an immunoglobulin single        variable domain comprises the amino acid residues at positions        66-94, CDR3 of an immunoglobulin single variable domain        comprises the amino acid residues at positions 95-102, and FR4        of an immunoglobulin single variable domain comprises the amino        acid residues at positions 103-113.    -   In the present application, however, unless indicated otherwise,        CDR sequences were determined according to Kontermann and Dübel        (Eds. 2010, Antibody Engineering, vol 2, Springer Verlag        Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to        this method, FR1 comprises the amino acid residues at positions        1-25, CDR1 comprises the amino acid residues at positions 26-35,        FR2 comprises the amino acids at positions 36-49, CDR2 comprises        the amino acid residues at positions 50-58, FR3 comprises the        amino acid residues at positions 59-94, CDR3 comprises the amino        acid residues at positions 95-102, and FR4 comprises the amino        acid residues at positions 103-113.

-   w) The Figures, Sequence Listing and the Experimental Part/Examples    are only given to further illustrate the invention and should not be    interpreted or construed as limiting the scope of the invention    and/or of the appended claims in any way, unless explicitly    indicated otherwise herein.

-   x) The half maximal inhibitory concentration (IC50) is a measure of    the effectiveness of a compound in inhibiting a biological or    biochemical function, e.g. a pharmacological effect. This    quantitative measure indicates how much of the ISV or Nanobody    (inhibitor) is needed to inhibit a given biological process (or    component of a process, i.e. an enzyme, cell, cell receptor,    chemotaxis, anaplasia, metastasis, invasiveness, etc) by half. In    other words, it is the half maximal (50%) inhibitory concentration    (IC) of a substance (50% IC, or IC50). The IC50 of a drug can be    determined by constructing a dose-response curve and examining the    effect of different concentrations of antagonist such as the ISV or    Nanobody of the invention on reversing agonist activity. IC50 values    can be calculated for a given antagonist such as the ISV or Nanobody    of the invention by determining the concentration needed to inhibit    half of the maximum biological response of the agonist.    -   The term half maximal effective concentration (EC50) refers to        the concentration of a compound which induces a response halfway        between the baseline and maximum after a specified exposure        time. In the present context it is used as a measure of a        polypeptide's, ISV's or Nanobody's potency. The EC50 of a graded        dose response curve represents the concentration of a compound        where 50% of its maximal effect is observed. Concentration is        preferably expressed in molar units.    -   In biological systems, small changes in ligand concentration        typically result in rapid changes in response, following a        sigmoidal function. The inflection point at which the increase        in response with increasing ligand concentration begins to slow        is the EC50. This can be determined mathematically by derivation        of the best-fit line. Relying on a graph for estimation is        convenient in most cases. In case the EC50 is provided in the        examples section, the experiments were designed to reflect the        KD as accurate as possible. In other words, the EC50 values may        then be considered as KD values. The term “average KD” relates        to the average KD value obtained in at least 1, but preferably        more than 1, such as at least 2 experiments. The term “average”        refers to the mathematical term “average” (sums of data divided        by the number of items in the data).    -   It is also related to IC50 which is a measure of a compound's        inhibition (50% inhibition). For competition binding assays and        functional antagonist assays, IC50 is the most common summary        measure of the dose-response curve. For agonist/stimulator        assays the most common summary measure is the EC50.

-   y) It must be noted that as used herein, the singular forms “a”,    “an”, and “the”, include plural references unless the context    clearly indicates otherwise. Thus, for example, reference to “a    reagent” includes one or more of such different reagents and    reference to “the method” includes reference to equivalent steps and    methods known to those of ordinary skill in the art that could be    modified or substituted for the methods described herein.    -   Unless otherwise indicated, the term “at least” preceding a        series of elements is to be understood to refer to every element        in the series. Those skilled in the art will recognize, or be        able to ascertain using no more than routine experimentation,        many equivalents to the specific embodiments of the invention        described herein. Such equivalents are intended to be        encompassed by the present invention.    -   The term “and/or” wherever used herein includes the meaning of        “and”, “or” and “all or any other combination of the elements        connected by said term”.    -   The term “about” or “approximately” as used herein means within        20%, preferably within 15%, more preferably within 10%, and most        preferably within 5% of a given value or range.    -   Throughout this specification and the claims which follow,        unless the context requires otherwise, the word “comprise”, and        variations such as “comprises” and “comprising”, will be        understood to imply the inclusion of a stated integer or step or        group of integers or steps but not the exclusion of any other        integer or step or group of integer or step. When used herein        the term “comprising” can be substituted with the term        “containing” or “including” or sometimes when used herein with        the term “having”.

The present invention relates to a polypeptide comprising at least afirst and at least one further immunoglobulin single variable domain(ISV), wherein said at least first ISV has high affinity for/binds tocluster of differentiation 3 (CD3) and said at least one further ISV hashigh affinity for/binds to an antigen on a target cell.

Typically, the multispecific polypeptides of the invention combine highaffinity antigen recognition on the target cell with T cell activation,resulting in an activation that is independent of the T cells' naturalspecificity. The mode of action of the binding molecules that binds bothto a cell surface molecule on a target cell such as a tumour antigen andto the T cell co-receptor CD3 is commonly known. Bringing a T cell inclose vicinity to a target cell, i.e., engaging said T cell results inkilling of the target cell by the T cell. In the present invention thisprocess is exploited in fighting against proliferative disease,inflammatory disease, infectious disease and autoimmune disease.Generally T cells are equipped with granules containing a deadlycombination of pore-forming proteins, called perforins, and celldeath-inducing proteases, called granzymes. Preferably, these proteinsare delivered into target cells via a cytolytic synapse that forms if Tcells are in close vicinity with a target cell that is aimed to bekilled. Normally, close vicinity between a T cell and a target cell isachieved by the T cell binding to an MHC/peptide complex using itsmatching T cell receptor. The polypeptides of the invention bring a Tcell into such close vicinity to a target cell in the absence of T cellreceptor/MHC interaction.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said polypeptide directs the T cell to the target cell.

With one arm (first ISV), the multispecific polypeptide has highaffinity for/binds to CD3, a protein component of the signal-transducingcomplex of the T cell receptor on T-cells. With another arm (second ISVand/or third ISV, etc.), the multispecific polypeptide recognizes, hashigh affinity for/binds an antigen(s) on target cells. Preferably, Tcell activation is only seen when the multispecific polypeptides arepresented to T cells on the surface of target cells. Antigen dependenceon target cells for activation results in a favourable safety profile.In an embodiment, the multispecific polypeptides transiently tether Tcells and target cells. Preferably, the multispecific polypeptide caninduce resting polyclonal T cells, such as CD4⁺ and/or CD8⁺ T cells intoactivation, for highly potent redirected lysis of target cells.Preferably, the T cell is directed to a next target cell after lysis ofthe first target cell.

Proteins and polypeptides that comprise or essentially consist of two ormore immunoglobulin single variable domains (such as at least twoimmunoglobulin single variable domains of the invention) will bereferred to herein as “multivalent” proteins or polypeptides or as“multivalent constructs”. Some non-limiting examples of such multivalentconstructs will become clear from the further description herein. Thepolypeptides of the invention are “multivalent”, i.e. comprising two ormore building blocks or ISVs of which at least the first building block,ISV or Nanobody and the second building block, ISV or Nanobody aredifferent, and directed against different targets, such as antigens orantigenic determinants. Polypeptides of the invention that contain atleast two building blocks, ISVs or Nanobodies, in which at least onebuilding block, ISV or Nanobody is directed against a first antigen(i.e., against the first target, such as e.g. CD3) and at least onebuilding block, ISV or Nanobody is directed against a second antigen(i.e., against the second target which is different from the firsttarget, such as e.g. a TAA, e.g. CD20 or HER2), will also be referred toas “multispecific” polypeptides of the invention, and the buildingblocks, ISVs or Nanobodies present in such polypeptides will also bereferred to herein as being in a “multivalent format” or “multispecificformat”. Thus, for example, a “bispecific” polypeptide of the inventionis a polypeptide that comprises at least one building block, ISV orNanobody directed against a first target (e.g. CD3) and at least onefurther building block, ISV or Nanobody directed against a second target(i.e., directed against a second target different from said firsttarget, such as e.g. a TAA, e.g. CD20 or HER2), whereas a “trispecific”polypeptide of the invention is a polypeptide that comprises at leastone building block, ISV or Nanobody directed against a first target(e.g., CD3), a second building block, ISV or Nanobody directed against asecond target different from said first target (e.g. a TAA, such as CD20or HER2) and at least one further building block, ISV or Nanobodydirected against a third antigen (i.e., different from both the firstand the second target, such as another TAA); etc. As will be clear fromthe description, the invention is not limited to bispecificpolypeptides, in the sense that a multispecific polypeptide of theinvention may comprise at least a first building block, ISV or Nanobodyagainst a first target, a second building block, ISV or Nanobody againsta second target and any number of building blocks, ISVs or Nanobodiesdirected against one or more targets, which may be the same or differentfrom the first and/or second target, respectively. The building blocks,ISVs or Nanobodies can optionally be linked via linker sequences.

The terms bispecific polypeptide, bispecific format, bispecificconstruct, bispecific Nanobody construct, bispecific and bispecificantibody are used interchangeably herein.

As will be clear from the further description above and herein, theimmunoglobulin single variable domains of the invention can be used as“building blocks” to form polypeptides of the invention, e.g., bysuitably combining them with other groups, residues, moieties or bindingunits, in order to form compounds or constructs as described herein(such as, without limitations, the bi-/tri-/tetra-/multivalent andbi-/tri-/tetra-/multispecific polypeptides of the invention describedherein) which combine within one molecule one or more desired propertiesor biological functions.

It will be appreciated (as is also demonstrated in the Example section)that the ISV binding CD3 and the ISV binding the antigen on a targetcell can be positioned in any order in the polypeptide of the invention.More particularly, in one embodiment, the ISV binding CD3 is positionedN-terminally and the ISV binding the antigen on a target cell ispositioned C-terminally. In another embodiment, the ISV binding theantigen on a target cell is positioned N-terminally and the ISV bindingCD3 is positioned C-terminally.

In a preferred aspect, the polypeptide of the invention comprises atleast a first, at least a second and at least a third immunoglobulinsingle variable domain (ISV), wherein said at least a first ISV has highaffinity for/binds to CD3; said at least a second ISV has high affinityfor/binds to a first antigen on a target cell, and said at least a thirdISV has high affinity for/binds to a second antigen on a target cell,wherein said second antigen is different from said first antigen. Saidfirst antigen and said second antigen can be on the same or on differenttarget cells.

It will be appreciated (as is also demonstrated in the Example section)that the ISV binding CD3 and the ISVs binding the first and secondantigen on a target cell can be positioned in any order in thepolypeptide of the invention. More particularly, in one embodiment, theISV binding CD3 is positioned N-terminally, the ISV binding the firstantigen on a target cell is positioned centrally and the ISV binding thesecond antigen on a target cell is positioned C-terminally. In anotherembodiment, the ISV binding CD3 is positioned N-terminally, the ISVbinding the second antigen on a target cell is positioned centrally andthe ISV binding the first antigen on a target cell is positionedC-terminally. In another embodiment, the ISV binding the first antigenon a target cell is positioned N-terminally, the ISV binding the secondantigen on a target cell is positioned centrally and the ISV binding CD3is positioned C-terminally. In another embodiment, the ISV binding thefirst antigen on a target cell is positioned N-terminally, the ISVbinding CD3 is positioned centrally and the ISV binding the secondantigen on a target cell is positioned C-terminally. In anotherembodiment, the ISV binding the second antigen on a target cell ispositioned N-terminally, the ISV binding CD3 is positioned centrally andthe ISV binding the first antigen on a target cell is positionedC-terminally. In another embodiment, the ISV binding the second antigenon a target cell is positioned N-terminally, the ISV binding the firstantigen on a target cell is positioned centrally and the ISV binding CD3is positioned C-terminally.

The invention further relates to compounds or constructs, and inparticular proteins or polypeptides that comprise or essentially consistof one or more ISVs or polypeptides of the invention, and optionallyfurther comprise one or more other groups, residues, moieties or bindingunits. As will become clear to the skilled person from the furtherdisclosure herein, such further groups, residues, moieties, bindingunits or amino acid sequences may or may not provide furtherfunctionality to the polypeptide of the invention (and/or to thecompound or construct in which it is present) and may or may not modifythe properties of the polypeptide of the invention.

The compounds, constructs or polypeptides of the invention can generallybe prepared by a method which comprises at least one step of suitablylinking the one or more immunoglobulin single variable domains of theinvention to the one or more further groups, residues, moieties orbinding units, optionally via one or more suitable linkers, so as toprovide the compound, construct or polypeptide of the invention.Polypeptides of the invention can also be prepared by a method whichgenerally comprises at least the steps of providing a nucleic acid thatencodes a polypeptide of the invention, expressing said nucleic acid ina suitable manner, and recovering the expressed polypeptide of theinvention. Such methods can be performed in a manner known per se, whichwill be clear to the skilled person, for example on the basis of themethods and techniques further described herein.

The process of designing/selecting and/or preparing a compound,construct or polypeptide of the invention, starting from an amino acidsequence of the invention, is also referred to herein as “formatting”said amino acid sequence of the invention; and an amino acid of theinvention that is made part of a compound, construct or polypeptide ofthe invention is said to be “formatted” or to be “in the format of” saidcompound, construct or polypeptide of the invention. Examples of ways inwhich an amino acid sequence of the invention can be formatted andexamples of such formats will be clear to the skilled person based onthe disclosure herein; and such formatted immunoglobulin single variabledomains or polypeptides form a further aspect of the invention.

For example, such further groups, residues, moieties or binding unitsmay be one or more additional immunoglobulin single variable domains,such that the compound or construct is a (fusion) protein or (fusion)polypeptide. In a preferred but non-limiting aspect, said one or moreother groups, residues, moieties or binding units are immunoglobulinsequences. Even more preferably, said one or more other groups,residues, moieties or binding units are chosen from the group consistingof domain antibodies, immunoglobulin single variable domains that aresuitable for use as a domain antibody, single domain antibodies,immunoglobulin single variable domains (ISVs) that are suitable for useas a single domain antibody, “dAb”'s, immunoglobulin single variabledomains that are suitable for use as a dAb, or Nanobodies.Alternatively, such groups, residues, moieties or binding units may forexample be chemical groups, residues, moieties, which may or may not bythemselves be biologically and/or pharmacologically active. For example,and without limitation, such groups may be linked to the one or moreimmunoglobulin single variable domains or polypeptides of the inventionso as to provide a “derivative” of an ESV or polypeptide of theinvention, as further described herein.

Also within the scope of the present invention are compounds orconstructs, which comprise or essentially consist of one or morederivatives as described herein, and optionally further comprise one ormore other groups, residues, moieties or binding units, optionallylinked via one or more linkers. Preferably, said one or more othergroups, residues, moieties or binding units are immunoglobulin singlevariable domains. In the compounds or constructs described above, theone or more immunoglobulin single variable domains of the invention andthe one or more groups, residues, moieties or binding units may belinked directly to each other and/or via one or more suitable linkers orspacers. For example, when the one or more groups, residues, moieties orbinding units are immunoglobulin single variable domains, the linkersmay also be immunoglobulin single variable domains, so that theresulting compound or construct is a fusion protein or fusionpolypeptide.

In some embodiments, the polypeptides comprise at least two or moreimmunoglobulin single variable domains disclosed herein. In someembodiments, the polypeptides essentially consist of two or moreimmunoglobulin single variable domains disclosed herein. A polypeptidethat “essentially consists of” two or more immunoglobulin singlevariable domains, is a polypeptide that in addition to the two or moreimmunoglobulin single variable domains disclosed herein does not haveadditional immunoglobulin single variable domains. For instance, apolypeptide that essentially consists of two immunoglobulin singlevariable domains does not include any additional immunoglobulin singlevariable domains. However, it should be appreciated that a polypeptidethat essentially consists of two or more immunoglobulin single variabledomains may include additional functionalities, such as a label, atoxin, one or more linkers, a binding sequence, etc. These additionalfunctionalities include both amino acid based and non-amino acid basedgroups. In some embodiments, the polypeptides consist of one or moreimmunoglobulin single variable domains disclosed herein. It should beappreciated that the terms “polypeptide construct” and “polypeptide” canbe used interchangeably herein (unless the context clearly dictatesotherwise).

In some embodiments, the polypeptides include multivalent ormultispecific constructs comprising immunoglobulin single variabledomains disclosed herein. In some embodiments, the polypeptides compriseone or more antibody based-scaffolds and/or non-antibody based scaffoldsdisclosed herein. In some embodiments, the polypeptides comprise a serumbinding protein moiety. In some embodiments, the serum binding proteinmoiety is an immunoglobulin single variable domain. In some embodiments,the immunoglobulin single variable domain is a Nanobody®.

It wilt be appreciated that the order of the building blocks, such ase.g. a first building block, a second building block, a third buildingblock etc., on the polypeptide (orientation) can be chosen according tothe needs of the person skilled in the art, as well as the relativeaffinities which may depend on the location of these building blocks inthe polypeptide. Whether the polypeptide comprises a linker, is a matterof design choice. However, some orientations, with or without linkers,may provide preferred binding characteristics in comparison to otherorientations. For instance, the order of a first and a second buildingblock in the polypeptide of the invention can be (from N-terminus toC-terminus): (i) first building block (e.g. a first ISV such as a firstNanobody)-[linker]-second building block (e.g. a second ISV such as asecond Nanobody); or (ii) second building block (e.g. a second ISV suchas a second Nanobody)-[linker]-first building block (e.g. a first ISVsuch as a first Nanobody); (wherein the linker is optional). Allorientations are encompassed by the invention. Polypeptides that containan orientation of building blocks that provides desired (binding)characteristics can be easily identified by routine screening, forinstance as exemplified in the experimental section.

The first immunoglobulin single variable domain (ISV) of the polypeptideof the invention has high affinity for/binds to an effector cell,preferably the TCR complex of said effector cell, and even morepreferably CD3.

An effector cell is a cell comprising a TCR complex, preferably animmune cell, such as a T-helper cell, monocyte, macrophage, or dendriticcell, preferably a CD4⁺ T-helper cell (also known as CD4 cell, T-helpercell or T4 cell), more preferably a Cytotoxic T cell (also known asT_(C) cell, CTL or CD8⁺ T cells), Natural Killer T cells (NKT cells) orNatural Killer cells (NK cells). In some embodiments, the cell ispresent in vivo. In some embodiments, the cell is present in vitro. Theeffector cell of the invention relates in particular to mammalian cells,preferably to primate cells, and even more preferably to human cells.

As used herein, the terms “TCR complex” or “αβ TCR-CD3 complex” refersto the T cell receptor complex presented on the surface of T cells (seeKuhns et al. 2006, Immunity 24:133-139). The TCR complex is composed ofsix different type I single-spanning transmembrane proteins: the TCRαand TCRβ chains that form the TCR heterodimer responsible for ligandrecognition, and the non-covalently associated CD3γ, CD3δ, CD3ε and ζchains, which bear cytoplasmic sequence motifs that are phosphorylatedupon receptor activation and recruit a large number of signallingcomponents. Both α and β chains of the T cell receptor consist of aconstant domain and a variable domain. The sequences for the human CD3and the human TCRα/β constant domains are provided in Table A-10 (SEQ IDNOs: 291-296; cf. UniProtKB: CD3 delta: P04234, CD3 gamma: P09693, CD3epsilon: P07766, CD3 zeta: P20963, TCR alpha: P01848 and TCR beta:P01850).

In an embodiment, the present invention relates to a polypeptide asdescribed herein, wherein said first ISV binds to CD3γ (SEQ ID NO: 292),to CD3δ (SEQ ID NO: 291) and/or CD3ε (SEQ ID NO: 293) of the TCRcomplex, or polymorphic variants or isoforms thereof.

Alternatively, the present invention provides a polypeptide as describedherein, wherein said first ISV binds to CD3γ (SEQ ID NO: 379), to CD3δ(SEQ ID NO: 291) and/or CD3ε (SEQ ID NO: 380) of the TCR complex, orpolymorphic variants or isoforms thereof.

Isoforms are alternative protein sequences that can be generated fromthe same gene by a single or by the combination of biological eventssuch as alternative promoter usage, alternative splicing, alternativeinitiation and ribosomal frameshifting, all as known in the art.

“T cell activation” as used herein refers to one or more cellularresponse(s) of a T cell, e.g. a cytotoxic T cell, such as selected from:proliferation, differentiation, cytokine secretion, cytotoxic effectormolecule release, cytotoxic activity, expression of activation markers,and redirected target cell lysis. The polypeptides of the invention arecapable of inducing T cell activation. Suitable assays to measure T cellactivation are known in the art described herein, for instance asdescribed in WO 99/54440 or by Schlereth et al. 2005 (Cancer Immunol.Immunother. 20: 1-12), or as exemplified in the examples or below.

In an embodiment, the present invention relates to a polypeptide asdescribed herein, wherein said polypeptide induces T cell activation.Preferably, the polypeptide of the invention induces T cell activationonly when said second and/or further ISV is bound to an antigen on atarget cell.

In an embodiment, the present invention relates to a polypeptide asdescribed herein, wherein said T cell activation depends on presentingsaid polypeptide bound to said first antigen on a target cell to a Tcell.

T cell activation by the polypeptides of the invention can be monitoredby upregulation of CD69, CD25 and various cell adhesion molecules, denovo expression and/or release of cytokines (e.g., IFN-γ, TNF-α, IL-6,IL-2, IL-4 and IL-10), upregulation of granzyme and perforin expression,and/or cell proliferation, membrane Webbing, activation of procaspases 3and/or 7, fragmentation of nuclear DNA and/or cleavage of caspasesubstrate poly (ADPribose) polymerase. Preferably, redirected lysis oftarget cells by multispecific polypeptides is independent of T cellreceptor specificity, presence of MHC class I and/or P2 microglobulin,and/or of any co-stimulatory stimuli.

In an embodiment, the present invention relates to a polypeptide asdescribed herein, wherein said T cell activation is independent from MHCrecognition.

The polypeptides of the invention show redirected lysis in vitro withpreviously unstimulated peripheral polyclonal CD8⁺- and CD4⁺-positive Tcells. The redirected lysis of target cells via the recruitment of Tcells by the polypeptides of the invention involves cytolytic synapseformation and delivery of perforin and granzymes. Cell lysis by T cellshas been described, e.g. by Atkinson and Bleackley 1995 (Crit. Rev.Immunol 15(3-4):359-384). Preferably, the engaged T cells are capable ofserial target cell lysis, and are not affected by immune escapemechanisms interfering with peptide antigen processing and presentation,or clonal T cell differentiation (see, for example, WO 2007/042261). Invitro, redirected lysis is seen at low picomolar concentrations,suggesting that very low numbers of the polypeptides of the inventionneed to be bound to target cells for triggering T cells. As demonstratedin the examples, the low effector to target ratio might be indicativefor serial target cell lysis. Accordingly, the present invention relatesto potent polypeptides. Preferably, the polypeptide of the inventionmediates killing of target cells, e.g., cancer cells, such asstimulating T cells in pore-forming and delivering pro-apoptoticcomponents of cytotoxic T cell granules.

In an embodiment, the present invention relates to a polypeptide asdescribed herein, wherein said T cell activation causes one or morecellular response of said T cell, wherein said cellular response isselected from the group consisting of proliferation, differentiation,cytokine secretion, cytotoxic effector molecule release, cytotoxicactivity, expression of activation markers and redirected target celllysis.

As used herein, the term “potency” is a measure of the biologicalactivity of an agent, such as a polypeptide, ISV or Nanobody. Potency ofan agent can be determined by any suitable method known in the art, suchas for instance as described in the experimental section. Cell culturebased potency assays are often the preferred format for determiningbiological activity since they measure the physiological responseelicited by the agent and can generate results within a relatively shortperiod of time. Various types of cell based assays, based on themechanism of action of the product, can be used, including but notlimited to proliferation assays, cytotoxicity assays, cell killingassays, reporter gene assays, cell surface receptor binding assays, andassays to measure induction/inhibition of functionally essential proteinor other signal molecules (such as phosphorytated proteins, enzymes,cytokines, cAMP and the like), Ramos B-cell depletion model, T cellmediated tumour cell killing assay (for instance as set out in theExamples section), all well known in the art. Results from cell basedpotency assays can be expressed as “relative potency” as determined bycomparison of the multispecific polypeptide of the invention to theresponse obtained for the corresponding reference monovalent ISV, e.g. apolypeptide comprising only one ISV or one Nanobody, optionally furthercomprising an irrelevant Nanobody (cf. experimental section).

In an embodiment, the present invention relates to a polypeptide asdescribed herein, wherein said T cell activation causes inhibition of anactivity of said target cell, such as to delay or minimize the spread ofthe target cell, to inhibit or delay growth and/or proliferation of thetarget cell, and/or to kill the target cell (e.g., cause regression ofthe disorder and/or symptoms) by more than about 10%, such as 20%, 30%,or 40% or even more than 50%, such as more than 60%, such as 70%, 80%,or even more than 90%, such 100%.

The first building block, ISV, Nanobody or VHH of the invention has ahigh affinity for its target, i.e. CD3. The first building block, ISV orNanobody of the invention may for example be directed against anantigenic determinant, epitope, part, domain, subunit or confirmation(where applicable) of said first target. The first building block, e.g.the first ISV, Nanobody or VHH, is preferably chosen for its highaffinity for its target per se, disregarding the influence of anyavidity effects.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said first ISV binds to CD3 with an average KD value ofbetween 100 nM and 10 pM, such as at an average KD value of 90 nM orless, even more preferably at an average KD value of 80 nM or less, suchas less than 70, 60, 50, 40, 30, 20, 10, 5 nM or even less, such as lessthan 4, 3, 2, or 1 nM, such as less than 500, 400, 300, 200, 100, 90,80, 70, 60, 50, 40, 30, 20 pM, or even less, such as less than 10 pM.Preferably, the KD is determined by Kinexa or SPR, for instance asdetermined by a Protean. For instance, said KD is determined as set outin the Examples section.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said first ISV has a high affinity when measured as amonovalent. Preferably said average KD is measured by surface plasmonresonance (SPR) on recombinant protein.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said polypeptide has a dissociation constant (K_(b)) to(or for binding) said CD3 selected from the group consisting of: at mostabout 10⁻⁵M, at most about 10⁻⁶M, at most about 10⁻⁷M, at most about10⁻⁸ M, at most about 10⁻⁹ M, at most about 10⁻¹⁰M, at most about 10⁻¹¹M, and at most about 10⁻¹²M, preferably as measured by surface plasmonresonance.

The present invention also relates to a polypeptide as described herein,wherein said first ISV binds to said CD3 with an EC50 value of between100 nM and 1 pM, such as at an average EC50 value of 100 nM or less,even more preferably at an average EC50 value of 90 nM or less, such asless than 80, 70, 60, 50, 40, 30, 20, 10, 5 nM or even less, such asless than 4, 3, 2, or 1 nM or even less, such as less than 500, 400,300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 pM, or even less,such as less than 4 pM.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said average KD is determined by FACS, Biacore, ELISA,on a monovalent first ISV, such as a Nanobody, or a polypeptidecomprising a monovalent first ISV, such as a Nanobody, for instance saidEC50 is determined as set out in the Examples section.

It has been shown in the examples that the KD correlates well with theEC50.

In an embodiment, the present invention relates to a polypeptide asdescribed herein, wherein said polypeptide has an on rate constant (Kon)to (or for binding) said CD3 selected from the group consisting of atleast about 10² M⁻¹s⁻¹, at least about 10³ M⁻¹s⁻¹, at least about 10⁴M⁻¹s⁻¹, at least about 10⁵ M⁻¹s⁻¹, at least about 10⁶ M⁻¹s⁻¹, 10⁷M⁻¹s⁻¹, at least about 10⁸ M⁻¹s⁻¹, at least about 10⁹ M⁻¹s⁻¹, and atleast about 10¹⁰ M⁻¹s⁻¹, preferably as measured by surface plasmonresonance or as performed in the examples section.

In an embodiment the present invention relates to a polypeptide asdescribed herein, wherein said polypeptide has an off rate constant(Koff) to (or for binding) said CD3 selected from the group consistingof at most about 10³s⁻¹, at most about 10⁻⁴s⁻¹, at most about 10⁻⁵s⁻¹,at most about 10⁻⁶s⁻¹, at most about 10⁻⁷s⁻¹, at most about 10⁻⁸s⁻¹, atmost about 10⁻⁹s⁻¹, and at most about 10⁻¹⁰s⁻¹, preferably as measuredby surface plasmon resonance or as performed in the examples section.

Amino acid sequence modifications of the binding molecules, ISVs, orpolypeptides described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody or ISV. Amino acid sequence variants of thebinding molecules, ISVs, or polypeptides are prepared by introducingappropriate nucleotide changes into the binding molecules, ISVs, orpolypeptides nucleic acid, or by peptide synthesis.

Such modifications include, for example, deletions from, and/orinsertions into, and/or substitutions of, residues within the amino acidsequences of the binding molecules, ISVs, or polypeptides. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also may alterpost-translational processes of the binding molecules, such as changingthe number or position of glycosylation sites. Preferably, 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids may be substituted in a CDR, while 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or25 amino acids may be substituted in the framework regions (FRs). Thesubstitutions are preferably conservative substitutions as describedherein. Additionally or alternatively, 1, 2, 3, 4, 5, or 6 amino acidsmay be inserted or deleted in each of the CDRs (of course, dependent ontheir length), while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or 25 amino acids may be inserted or deleted in eachof the FRs.

A useful method for identification of certain residues or regions of thebinding molecules, ISVs or polypeptides, that are preferred locationsfor mutagenesis is called “alanine scanning mutagenesis” as described byCunningham and Wells 1989 (Science 244: 1081-1085). Here, a residue orgroup of target residues within the binding molecule is/are identified(e.g. charged residues such as Arg, Asp, His, Lys, and Glu) and replacedby a neutral or negatively charged amino acid (most preferably alanineor polyalanine) to affect the interaction of the amino acids with theepitope. Those amino acid locations demonstrating functional sensitivityto the substitutions then are refined by introducing further or othervariants at, or for, the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se needs not to be predetermined. Forexample, to analyze the performance of a mutation at a given site, alascanning or random mutagenesis is conducted at a target codon or regionand the expressed binding molecule variants are screened for the desiredactivity.

Preferably, amino acid sequence insertions include amino- and/orcarboxyl-terminal fusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8,9 or 10 residues to polypeptides containing a hundred or more residues.

Another type of variant is an amino acid substitution variant. Thesevariants have preferably at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 aminoacid residues in the binding molecule, ISV or polyptide replaced by adifferent residue. The sites of greatest interest for substitutionmutagenesis include the CDRs, in particular the hypervariable regions,but FR alterations are also contemplated. For example, if a CDR sequenceencompasses 6 amino acids, it is envisaged that one, two or three ofthese amino acids are substituted. Similarly, if a CDR sequenceencompasses 15 amino acids it is envisaged that one, two, three, four,five or six of these amino acids are substituted.

Generally, if amino acids are substituted in one or more or all of theCDRs, it is preferred that the then-obtained “substituted” sequence isat least 60%, more preferably 65%, even more preferably 70%,particularly preferably 75%, more particularly preferably 80% or evenmore than 90% identical to the “original” CDR sequence. This means thatit is dependent of the length of the CDR to which degree it is identicalto the “substituted” sequence. For example, a CDR having 5 amino acidsis preferably 80% identical to its substituted sequence in order to haveat least one amino acid substituted. Accordingly, the CDRs of thebinding molecule may have different degrees of identity to theirsubstituted sequences, e.g., CDR1 may have 80%, while CDR3 may have 90%.

Preferred substitutions (or replacements) are conservativesubstitutions. However, any substitution (including non-conservativesubstitution or one or more from the “exemplary substitutions” listed inTable B-1 below) is envisaged as long as the polypeptide retains itscapability to bind to CD3 present on a T cell via the first ISV and to afirst antigen on a target cell via the second ISV and/or its CDRs havean identity to the then substituted sequence (at least 60%, morepreferably 65%, even more preferably 70%, particularly preferably 75%,more particularly preferably 80% identical to the “original” CDRsequence).

Conservative substitutions are shown in Table B-1 below.

TABLE B-1 Amino Acid Substitutions Original Exemplary SubstitutionsPreferred Substitutions Ala (A) val, leu, ile val Arg (R) lys, gln, asnlys Asn (N) gln, his, asp, lys, arg gln Asp (D) glu, asn glu Cys (C)ser, ala ser Gln (Q) asn, glu asn Glu (E) asp, gln asp Gly (G) ala alaHis (H) asn, gln, lys, arg arg Ile (I) leu, val, met, ala, phe leu Leu(L) norleucine, ile, val, met, ala ile Lys (K) arg, gln, asn arg Met (M)leu, phe, ile leu Phe (F) leu, val, ile, ala, tyr tyr Pro (P) ala alaSer (S) thr thr Thr (T) ser ser Trp (W) tyr, phe tyr Tyr (Y) trp, phe,thr, ser phe Val (V) ile, leu, met, phe, ala leu

Sequence analysis further revealed that there are only a limited numberof sequence variations in the CDRs (cf. Example 4.2 and Tables A-1 toA-6).

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 81-100; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 81 or with any of SEQ ID NOs: 81-100; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 101-122; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 101 or with any of SEQ ID NOs: 101-122; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 123-143; and        -   (f) amino acid sequences that have 3, 2, or 1 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 123 or            with any of SEQ ID NOs: 123-143.

Further preferred CDR sequences are depicted in Table A-8.

Generally, the combinations of CDR's listed in Table A-8 (i.e. thosementioned on the same line in Table A-4) are preferred. Thus, it isgenerally preferred that, when a CDR in an ISV is a CDR sequencementioned in Table A-8 or suitably chosen from the group consisting ofCDR sequences that have 4, 3, 2 or only 1 amino acid difference(s) witha CDR sequence listed in Table A-8, that at least one and preferablyboth of the other CDR's are suitably chosen from the CDR sequences thatbelong to the same combination in Table A-8 (i.e. mentioned on the sameline in Table A-8) or are suitably chosen from the group consisting ofCDR sequences that have 4, 3, 2 or only 1 amino acid difference(s) withthe CDR sequence(s) belonging to the same combination.

Sequence analysis of the resulting binders further resulted in theidentification of 6 distinct clusters. Corresponding alignments areprovided (see Table A-1, Table A-2, Table A-3, Table A-4, Table A-S andTable A-6). Clustering was based on sequence similarities anddifferences in CDR2 and CDR3. Cluster A is the most prominent comprising50 clones (SEQ ID NO:s 1-50), cluster B and cluster D are eachrepresented by only 1 clone (SEQ ID NO: 51 and SEQ ID NO: 52,respectively), cluster C comprises 4 clones (SEQ ID NO:s 53-56), clusterE comprises 9 clones (SEQ ID NO:s 57-65) and cluster F comprises 15clones (SEQ ID NO:s 66-80). The clustering based on the structuralsimilarities and differences in the amino acid sequence translated intofunctional similarities and differences as revealed by the examples.Representatives of all clusters were isolated based on high affinitybinding to CD3 (Examples 3 & 4) and human T cell activation (Example4.2). In general cluster A representatives demonstrated the best EC50values. Although cluster C representatives had somewhat less favourableEC50 values than cluster B representatives, cluster C representativeshad lower IC50 values in a flow cytometry based T cell mediated Ramoskilling assay (cf. Example 10).

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which CDR1 is chosen from thegroup consisting of

-   -   (a) SEQ ID NO: 81; and    -   (b) amino acid sequences that have 1, 2, 3 or 4 amino acid        difference(s) with SEQ ID NO: 81, wherein        -   at position 1 the G has been changed into R;        -   at position 3 the T has been changed into A;        -   at position 4 the Y has been changed into F;        -   at position 8 the S has been changed into G; and/or        -   at position 10 the G has been changed into A.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which CDR2 is chosen from thegroup consisting of

-   -   (a) SEQ ID NO: 101; and    -   (b) amino acid sequences that have 1, 2, 3 or 4 amino acid        difference(s) with SEQ ID NO: 101, wherein        -   at position 3 the V has been changed into T or A;        -   at position 5 the S has been changed into T;        -   at position 6 the G has been changed into D or E; and/or        -   at position 9 the T has been changed into S, A or P.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which CDR3 is chosen from thegroup consisting of

-   -   (a) SEQ ID NO: 123; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NO: 123, wherein        -   at position 2 the I has been changed into T;        -   at position 9 the I has been changed into V; and/or        -   at position 10 the A has been changed into P.

In an embodiment, the invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 81-87; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 81; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 101-109; and        -   (d) amino add sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 101; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 123-127; and        -   (f) amino acid sequences that have 3, 2, or 1 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 123.

In an embodiment, the invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which: CDR1 is represented bySEQ ID NO: 81, CDR2 is represented by SEQ ID NO: 101, and CDR3 isrepresented by SEQ ID NO: 123.

Nanobodies belonging to cluster B are represented by 1 clone.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NO: 88; and        -   (b) amino acid sequences that have 1, 2, 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 88; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NO: 110; and        -   (d) amino acid sequences that have 1, 2, 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 110; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NO: 128; and        -   (f) amino acid sequences that have 1, 2, or 3 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 128.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which: CDR1 is represented bySEQ ID NO: 88, CDR2 is represented by SEQ ID NO: 110, and CDR3 isrepresented by SEQ ID NO: 128.

Nanobodies of cluster C show very limited sequence variability in theCDRs.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which CDR2 is chosen from thegroup consisting of

-   -   (a) SEQ ID NO: 112; and    -   (b) amino acid sequence that has 1 amino acid difference with        SEQ ID NO: 112, wherein        -   at position 2 the V has been changed into A.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NO: 90; and        -   (b) amino acid sequences that have 1, 2, 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 90; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 112-113; and        -   (d) amino acid sequences that have 1, 2, 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 112; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NO: 130; and        -   (f) amino acid sequences that have 1, 2, or 3 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 130.

In an aspect, the present invention relates to a polypeptide asdescribed herein, in which said first ISV essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which: CDR1 isrepresented by SEQ ID NO: 90, CDR2 is represented by SEQ ID NO: 112, andCDR3 is represented by SEQ ID NO: 130.

Nanobodies belonging to cluster D are represented by 1 clone.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NO: 89; and        -   (b) amino acid sequences that have 1, 2, 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 89; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NO: 111; and        -   (d) amino acid sequences that have 1, 2, 3, or 4 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 111; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NO: 129; and        -   (f) amino acid sequences that have 1, 2, or 3 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 129.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which: CDR1 is represented bySEQ ID NO: 89, CDR2 is represented by SEQ ID NO: 111, and CDR3 isrepresented by SEQ ID NO: 129.

Cluster E comprises 9 clones.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which CDR1 is chosen from thegroup consisting of

-   -   (a) SEQ ID NO: 91; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid(s)        difference with SEQ ID NO: 91, wherein        -   at position 6 the R has been changed into N or T;        -   at position 7 the N has been changed into H; and/or        -   at position 8 the M has been changed into T.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which CDR2 is chosen from thegroup consisting of

-   -   (a) SEQ ID NO: 114; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid(s)        difference with SEQ ID NO: 114, wherein        -   at position 1 the R has been changed into Q;        -   at position 3 the T has been changed into S; and/or        -   at position 7 the D has been changed into A or K.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which CDR3 is chosen from thegroup consisting of

-   -   (a) SEQ ID NO: 131; and    -   (b) amino acid sequences that have 1 or 2 amino acid(s)        difference with SEQ ID NO: 131, wherein        -   at position 2 the S has been changed into R; and/or        -   at position 6 the S has been changed into V.

In an embodiment, the invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity so determiningregions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 91-93; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 91; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 114-117; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 114; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 131-133; and        -   (f) amino acid sequences that have 3, 2, or 1 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 131.

In an embodiment, the invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which: CDR1 is represented bySEQ ID NO: 91, CDR2 is represented by SEQ ID NO: 114, and CDR3 isrepresented by SEQ ID NO: 131.

Nanobodies belonging to cluster F are represented by 15 clones.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which CDR1 is chosen from thegroup consisting of

-   -   (a) SEQ ID NO: 94; and    -   (b) amino acid sequences that have 1, 2, 3 or 4 amino acid(s)        difference with SEQ ID NO: 94, wherein        -   at position 3 the S has been changed into T, A or G;        -   at position 5 the N has been changed into S;        -   at position 6 the M has been changed into T or A; and/or        -   at position 9 the L has been changed into M.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which CDR2 is chosen from thegroup consisting of

-   -   (a) SEQ ID NO: 118; and    -   (b) amino acid sequences that have 1, 2, 3 or 4 amino acid(s)        difference with SEQ ID NO: 118, wherein        -   at position 2 the H has been changed into V;        -   at position 5 the S has been changed into H or A;        -   at position 8 the N has been changed into S; and/or        -   at position 10 the Y has been changed into F.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which CDR3 is chosen from thegroup consisting of

-   -   (a) SEQ ID NO: 134; and    -   (b) amino acid sequences that have 1, 2, 3 or 4 amino acid(s)        difference with SEQ ID NO: 134, wherein        -   at position 6 the A has been changed into S or D;        -   at position 7 the F has been changed into Y or A;        -   at position 8 the R has been changed into H;        -   at position 9 the S has been changed into A;        -   at position 11 the G has been changed into D, T, N, 5, K or            R; and/or        -   at position 14 the V has been changed into I.

In an embodiment, the invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 94-100; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 94; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 118-122; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 118; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 134-143; and        -   (f) amino acid sequences that have 3, 2, or 1 amino acid(s)            difference with the amino acid sequence of SEQ ID NO: 134.

In an embodiment, the invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which: CDR1 is represented bySEQ ID NO: 94, CDR2 is represented by SEQ ID NO: 118, and CDR3 isrepresented by SEQ ID NO: 134.

The second immunoglobulin single variable domain (ISV) of thepolypeptide of the invention has a high affinity for/binds to an antigenon a target cell, preferably a cancer cell. A “target cell” as referredto herein, is a cell that presents a particular antigen on its surface.In a preferred aspect, the “target cell” is a cancer cell.

The membrane (also called plasma membrane or phospholipid bilayer)surrounds the cytoplasm of a cell, which is the outer boundary of thecell, i.e. the membrane is the surface of the cell. This membrane servesto separate and protect a cell from its surrounding environment and ismade mostly from a double layer of phospholipids. Embedded within thismembrane is a variety of protein molecules, such as channels, pumps andcellular receptors. Since the membrane is fluid, the protein moleculescan travel within the membrane. The term “antigen on a target cell” asused herein denotes a molecule, which is displayed on the surface of acell. In most cases, this molecule will be located in or on the plasmamembrane of the cell such that at least part of this molecule remainsaccessible from outside the cell in tertiary form. A non-limitingexample of a cell surface molecule, which is located in the plasmamembrane, is a transmembrane protein comprising, in its tertiaryconformation, regions of hydrophilicity and hydrophobicity. Here, atleast one hydrophobic region allows the cell surface molecule to beembedded, or inserted in the hydrophobic plasma membrane of the cellwhile the hydrophilic regions extend on either side of the plasmamembrane into the cytoplasm and extracellular space, respectively.

Said antigen can be any target on a cell, e.g. a tumour antigen. In apreferred embodiment, said antigen is specific for said target cell,e.g. cancer cell, such as a tumour associated antigen (TAA) on saidcancer cell.

The term “tumour antigen” as used herein may be understood as thoseantigens that are presented on tumour cells. These antigens can bepresented on the cell surface with an extracellular part, which is oftencombined with a transmembrane and cytoplasmic part of the molecule.These antigens can sometimes be presented only by tumour cells and neverby a normal or healthy cell. Tumour antigens can be exclusivelyexpressed on tumour cells or might represent a tumour specific mutationcompared to normal cells. In this case, they are called tumour-specificantigens. However, this will not be the case generally. More common areantigens that are presented by tumour cells and normal cells, and theyare called “tumour-associated antigens (TAA)”. These tumour-associatedantigens can be overexpressed on tumour cells compared to normal cellsor are better accessible for antibody binding in tumour cells due to theless compact structure of the tumour tissue compared to normal tissue.TAA are preferably antigens that are expressed on cells of particulartumours, but that are preferably not expressed in normal cells. Often,TAA are antigens that are normally expressed in cells only at particularpoints in an organism's development (such as during fetal development)and that are being inappropriately expressed in the organism at thepresent point of development, or are antigens not expressed in normaltissues or cells of an organ now expressing the antigen.

In an embodiment, said first antigen on a target cell is a tumourantigen, preferably a tumour associated antigen (TM).

In an embodiment, said second antigen on a target cell is a tumourantigen, preferably a tumour associated antigen (TAA).

In an embodiment, said antigen is present more abundantly on a cancercell than on a normal cell. The antigen on a target cell is preferably atumor-associated antigen (TAA). Preferred TAA include MART-1,carcinoembryonic antigen (“CEA”), gp100, MAGE-1, HER-2, CD20, Lewis^(Y)antigens, Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP),Epidermal Growth Factor Receptor (EGFR), Fibroblast Activation Protein(FAP), CD19 and CD33.

Cell surface antigens that are preferentially expressed on AML LSCcompared with normal hematopoietic stem cells, and thus preferred asTAA, include CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3 andCD25.

Other tumor-associated antigens suitable as an antigen on a target cellfor binding by the second ISV within the polypeptides of the inventioninclude: TAG-72, Ep-CAM, PSMA, PSA, glycolipids such as GD2 and GD3.

The TAA of the invention include also hematopoietic differentiationantigens, i.e. glycoproteins usually associated with clusterdifferentiation (CD) grouping, such as CD4, CD5, CD19, CD20, CD22, CD33,CD36, CD45, CD52, CD69 and CD147; growth factor receptors, includingHER2, ErbB3 and ErbB4; Cytokine receptors, including Interleukin-2receptor gamma chain (CD132 antigen), interleukin-10 receptor alphachain (IL-10R-A), Interleukin-10 receptor beta chain (IL-10R-B),Interleukin-12 receptor beta-1 chain (IL-12R-beta1), Interleukin-12receptor beta-2 chain (IL-12 receptor beta-2), Interleukin-13 receptoralpha-1 chain (IL-13R-alpha-1) (CD213a1 antigen), Interleukin-13receptor alpha-2 chain (Interleukin-13 binding protein), Interleukin-17receptor (IL-17 receptor), Interleukin-17B receptor (IL-17B receptor),Interleukin 21 receptor precursor (IL-21R), Interleukin-1 receptor typeI (IL-1R-1) (CD121a), Interleukin-1 receptor type II (IL-1R-beta)(CDw121b), Interleukin-1 receptor antagonist protein (IL-1ra),Interleukin-2 receptor alpha chain (CD25 antigen), Interleukin-2receptor beta chain (CD122 antigen), Interleukin-3 receptor alpha chain(IL-3R-alpha) (CD123 antigen); as well as others, such as CD30, 1L23R,IGF-1R, 115R, IgE, CD248 (endosialin), CD44v6, gpA33, Ron, Trop2, PSCA,claudin 6, claudin 18.2, CLEC12A, CD38, ephA2, c-Met, CD56, MUC16,EGFRvIII, AGS-16, CD27L, Nectin-4, SLITRK6, mesothelin, folate receptor,tissue factor, axl, glypican-3, CA9, Cripto, CD138, CD37, MUC1, CD70,gastrin releasing peptide receptor, PAP, CEACAM5, CEACAM6, CXCR7,N-cadherin, FXYD2 gamma a, CD21, CD133, Na/K-ATPase, mIgM(membrane-bound IgM), mIgA (membrane-bound IgA), Mer, Tyro2, CD120,CD95, CA 195, DR5, DR6, DcR3 and CAIX.

Accordingly the present invention relates to a polypeptide as describedherein, wherein said TAA is chosen from the group consisting ofMelanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), EpidermalGrowth Factor Receptor (EGFR), Fibroblast Activation Protein (FAP),MART-1, carcinoembryonic antigen (“CEA”), gp100, MAGE-1, HER-2,Lewis^(Y) antigens, CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3,CD25, TAG-72, Ep-CAM, PSMA, PSA, GD2, GD3, CD4, CD5, CD19, CD20, CD22,CD33, CD36, CD45, CD52, CD147; growth factor receptors, including ErbB3and ErbB4; Cytokine receptors, including Interleukin-2 receptor gammachain (CD132 antigen), Interleukin-10 receptor alpha chain (IL-10R-A),Interleukin-10 receptor beta chain (IL-10R-B), Interleukin-12 receptorbeta-1 chain (IL-12R-beta1), Interleukin-12 receptor beta-2 chain (IL-12receptor beta-2), Interleukin-13 receptor alpha-1 chain (IL-13R-alpha-1)(CD213a1 antigen), Interleukin-13 receptor alpha-2 chain (Interleukin-13binding protein), Interleukin-17 receptor (IL-17 receptor),Interleukin-178 receptor (IL-17B receptor), Interleukin 21 receptorprecursor (IL-21R), Interleukin-1 receptor type I (IL-1R-1) (CD121a),Interleukin-1 receptor type I1 (IL-1R-beta) (CDw121b), Interleukin-1receptor antagonist protein (IL-1ra), Interleukin-2 receptor alpha chain(CD25 antigen), Interleukin-2 receptor beta chain (CD122 antigen),Interleukin-3 receptor alpha chain (IL-3R-alpha) (CD123 antigen), CD30,IL23R, IGF-1R, IL5R, IgE, CD248 (endosialin), CD44v6, gpA33, Ron, Trop2,PSCA, claudin 6, claudin 18.2, CLEC12A, CD38, ephA2, c-Met, CD56, MUC16,EGFRvIII, AGS-16, CD27L, Nectin-4, SLITRK6, mesothelin, folate receptor,tissue factor, axl, glypican-3, CA9, Cripto, CD138, CD37, MUC1, CD70,gastrin releasing peptide receptor, PAP, CEACAM5, CEACAM6, CXCR7,N-cadherin, FXYD2 gamma a, CD21, CD133, Na/K-ATPase, mIgM(membrane-bound IgM), mIgA (membrane-bound IgA), Mer, Tyro2, CD120,CD95, CA 195, DR5, DR6, DcR3 and CAIX, and related polymorphic variantsand isoforms, preferably said TAA is CD20 (UniProt 11836), HER2 (UniprotP04626), polymorphic variants and/or isoforms thereof.

The second building block, ISV, Nanobody or VHH of the invention has ahigh affinity for its antigen. The second building block, ISV orNanobody of the invention may, for example, be directed against anantigenic determinant, epitope, part, domain, subunit or confirmation(where applicable) of said antigen on a target cell.

The target cell of the invention relates in particular to mammaliancells, and preferably to primate cells and even more preferably to humancells. The target cell is preferably a hyperproliferative cell such ase.g. a cancer cell.

The present invention relates to a polypeptide as described herein,wherein said second or further ISV binds to an antigen on a target cellwith an average KD value of between 100 nM and 10 pM, such as at anaverage KD value of 90 nM or less, even more preferably at an average KDvalue of 80 nM or less, such as less than 70, 60, 50, 40, 30, 20, 10, 5nM or even less, such as less than 4, 3, 2, or 1 nM, such as less than500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 pM, or even lesssuch as less than 10 pM. Preferably, the KD is determined by KinExA orSPR, for instance as determined or Proteon.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said second or further ISV has a high affinity for itsantigen when measured as a monovalent.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said average KD is measured by surface plasmon resonance(SPR) and/or KinExA or Proteon, for instance on recombinant protein,such as described in the Examples section.

The present invention also relates to a polypeptide as described herein,wherein said second or further ISV binds to an antigen on a target cellwith an EC50 value of between 100 nM and 1 pM, such as at an averageEC50 value of 100 nM or less, even more preferably at an average EC50value of 90 nM or less, such as less than 80, 70, 60, 50, 40, 30, 20,10, 5 nM or even less, such as less than 4, 3, 2, or 1 nM or even lesssuch as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30,20, 10, 5 pM, or even less such as less than 4 pM.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said average EC50 is determined by FACS, or ELISA, on amonovalent second ISV, such as a Nanobody, or a polypeptide comprising amonovalent second ISV, such as a Nanobody.

It has been shown in the examples that the KD correlates well with theEC50.

Simultaneous targeting of multiple antigens can reduce the probabilityof generating tumour escape variants, because of which the therapeuticactivity of T cell engaging strategy is improved. The present inventionprovides multispecific polypeptides which comprise a CD3 ISV combinedwith immunoglobulin single variable domains against different (target)antigens (on a target cell) (cf. Example 19). Preferred combinations offirst and second antigens are provided below (it will be appreciatedthat the ISVs binding said antigens can be positioned in any order inthe polypeptide of the invention):

first antigen second antigen EGFR (OMIM: 131550) CD20 (OMIM: 112210)EGFR (OMIM: 131550) CEA (OMIM: 114890) EGFR (OMIM: 131550) HER2 (OMIM:164870) HER2 (OMIM: 164870) CD20 (OMIM: 112210) HER2 (OMIM: 164870) CEA(OMIM: 114890) CD20 (OMIM: 112210) CEA (OMIM: 114890)

Similarly, simultaneous targeting of multiple epitopes, antigenicdeterminants, parts, domains, subunit or conformation of a protein orantigen on a target cell can reduce the probability of generating tumourescape variants, because of which the therapeutic activity of T cellengaging strategy is improved (cf. Example 20). The present inventionprovides polypeptides which comprise a anti-CD3 ISV combined withimmunoglobulin single variable domains against different epitopes,antigenic determinants, parts, domains, subunit or conformation of anantigen on a target cell (also referred to as biparatopic constructs).Preferred combinations of first and second TAA ISVs are provided below(it will be appreciated that the ISVs binding said antigens can bepositioned in any order in the polypeptide of the invention):

TAA1 TAA2 ISV name SEQ ID NO: ISV name SEQ ID NO: EGFR-1 7D12 302 EGFR-29G08 299 HER2-1 5F07 297 HER2-2 47D05 298 CEA-1 CEA#1 300 CEA-2 CEA#5301

The polypeptides and compositions of the present invention can be usedfor the prevention and/or treatment of diseases and disorders of thepresent invention (herein also “diseases and disorders of the presentinvention”) which include, but are not limited to cancer. The term“cancer” refers to the pathological condition in mammals that istypically characterized by dysregulated cellular proliferation orsurvival. Examples of cancer include, but are not limited to,carcinomas, gliomas, mesotheliomas, melanomas, lymphomas, leukemias,adenocarcinomas: breast cancer, ovarian cancer, cervical cancer,glioblastoma, multiple myeloma (including monoclonal gammopathy ofundetermined significance, asymptomatic and symptomatic myeloma),prostate cancer, and Burkitt's lymphoma, head and neck cancer, coloncancer, colorectal cancer, non-small cell lung cancer, small cell lungcancer, cancer of the esophagus, stomach cancer, pancreatic cancer,hepatobiliary cancer, cancer of the gallbladder, cancer of the smallintestine, rectal cancer, kidney cancer, bladder cancer, prostatecancer, penile cancer, urethral cancer, testicular cancer, vaginalcancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenalcancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skincancer, retinoblastomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma,Kaposi's sarcoma, multicentric Castleman's disease or AIDS-associatedprimary effusion lymphoma, neuroectodermal tumors, rhabdomyosarcoma (seee.g., Cancer, Principles and practice (DeVita, et al. eds 1997) foradditional cancers); as well as any metastasis of any of the abovecancers, as well as non-cancer indications such as nasal polyposis; aswell as other disorders and diseases described herein.

For a general description of immunoglobulin single variable domains,reference is made to the further description below, as well as to theprior art cited herein. In this respect, it should however be noted thatthis description and the prior art mainly describes immunoglobulinsingle variable domains of the so-called “V_(H)3 class” (i.e.,immunoglobulin single variable domains with a high degree of sequencehomology to human germline sequences of the V_(H)3 class such as DP-47,DP-51 or DP-29), which form a preferred aspect of this invention. Itshould, however, be noted that the invention in its broadest sensegenerally covers any type of immunoglobulin single variable domains andfor example also covers the immunoglobulin single variable domainsbelonging to the so-called “V_(H)4 class” (i.e., immunoglobulin singlevariable domains with a high degree of sequence homology to humangermline sequences of the V_(H)4 class such as DP-78), as for exampledescribed in WO 07/118670.

Generally, immunoglobulin single variable domains (in particular V_(HH)sequences and sequence optimized immunoglobulin single variable domains)can in particular be characterized by the presence of one or more“Hallmark residues” (as described for example in Table B-2) in one ormore of the framework sequences (again as further described herein).

TABLE B-2 Hallmark Residues in VHHs Position Human V_(H)3 HallmarkResidues 11 L, V; predominantly L L, S, V, M, W, F, T, Q, E, A, R, G, K,Y, N, P, I; preferably L 37 V, I, F; usually V F⁽¹⁾, Y, V, L, A, H, S,I, W, C, N, G, D, T, P, preferably F⁽¹⁾ or Y  44⁽⁸⁾ G E⁽³⁾, Q⁽³⁾, G⁽²⁾,D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably G⁽²⁾, E⁽³⁾ or Q⁽³⁾;most preferably G⁽²⁾ or Q⁽³⁾.  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, P, H, F, G, Q, S, E,T, Y, C, I, D, V; preferably L⁽²⁾ or R⁽³⁾  47⁽⁸⁾ W, Y F⁽¹⁾, L⁽¹⁾ or W⁽²⁾G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D; preferably W⁽²⁾, L⁽¹⁾or F⁽¹⁾ 83 R or K; usually R R, K⁽⁵⁾, T, E⁽⁵⁾, Q, N, S, I, V, G, M, L,A, D, Y, H; preferably K or R; most preferably K 84 A, T, D;predominantly A P⁽⁵⁾, S, H, L, A, V, I, T, F, D, R, Y, N, Q, G, E;preferably P 103  W W⁽⁴⁾, R⁽⁶⁾, G, S, K, A, M, Y, L, F, T, N, V, Q,P⁽⁶⁾, E, C; preferably W 104  G G, A, S, T, D, P, N, E, C, L; preferablyG 108  L, M or T; predominantly L Q, L⁽⁷⁾, R, P, E, K, S, T, M, A, H;preferably Q or L⁽⁷⁾ Notes: ⁽¹⁾In particular, but not exclusively, incombination with KERE or KQRE at positions 43-46. ⁽²⁾Usually as GLEW atpositions 44-47. ⁽³⁾Usually as KERE or KQRE at positions 43-46, e.g. asKEREL, KEREF, KQREL, KQREF, KEREG, KQREW or KQREG at positions 43-47.Alternatively, also sequences such as TERE (for example TEREL), TQRE(for example TQREL), KECE (for example KECEL or KECER), KQCE (forexample KQCEL), RERE (for example REREG), RQRE (for example RQREL, RQREFor RQREW), QERE (for example QEREG), QQRE, (for example QQREW, QQREL orQQREF), KGRE (for example KGREG), KDRE (for example KDREV) are possible.Some other possible, but less preferred sequences include for exampleDECKL and NVCEL. ⁽⁴⁾With both GLEW at positions 44-47 and KERE or KQREat positions 43-46. ⁽⁵⁾Often as KP or EP at positions 83-84 of naturallyoccurring V_(HH) domains. ⁽⁶⁾In particular, but not exclusively, incombination with GLEW at positions 44-47. ⁽⁷⁾With the proviso that whenpositions 44-47 are GLEW, position 108 is always Q in (non-humanized)V_(HH) sequences that also contain a W at 103. ⁽⁸⁾The GLEW group alsocontains GLEW-like sequences at positions 44-47, such as for exampleGVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER andELEW.

The immunoglobulins of the invention may also contain a C-terminalextension (X)n (in which n is 1 to 10, preferably 1 to 5, such as 1, 2,3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an(preferably naturally occurring) amino acid residue that isindependently chosen, and preferably independently chosen from the groupconsisting of alanine (A), glycine (6), valine (V), leucine (1) orisoleucine (I)), for which reference is made to WO 12/175741 and WO15/060643.

Apart from this and/or in addition, the immunoglobulin of the inventionmay have certain preferred amino acid residues at positions 11, 89, 110and/or 112 as is described in further detail in WO 15/060643 (which isincorporated herein as reference).

Again, such immunoglobulin single variable domains may be derived in anysuitable manner and from any suitable source, and may for example benaturally occurring V_(HH) sequences (i.e., from a suitable species ofCamelid, e.g., llama) or synthetic or semi-synthetic VHs or VLs (e.g.,from human). Such immunoglobulin single variable domains may include“humanized” or otherwise “sequence optimized” VHHs, “camelized”immunoglobulin sequences (and in particular camelized heavy chainvariable domain sequences, i.e., camelized VHs), as well as human VHs,human VLs, camelid VHHs that have been altered by techniques such asaffinity maturation (for example, starting from synthetic, random ornaturally occurring immunoglobulin sequences), CDR grafting, veneering,combining fragments derived from different immunoglobulin sequences, PCRassembly using overlapping primers, and similar techniques forengineering immunoglobulin sequences well known to the skilled person;or any suitable combination of any of the foregoing as further describedherein. As mentioned herein, a particularly preferred class ofimmunoglobulin single variable domains of the invention comprisesimmunoglobulin single variable domains with an amino acid sequence thatcorresponds to the amino acid sequence of a naturally occurring V_(HH)domain, but that has been “humanized”, i.e. by replacing one or moreamino acid residues in the amino acid sequence of said naturallyoccurring V_(HH) sequence (and in particular in the framework sequences)by one or more of the amino acid residues that occur at thecorresponding position(s) in a V_(H) domain from a conventional 4-chainantibody from a human being (e.g. indicated above). This can beperformed in a manner known per se, which will be clear to the skilledperson, for example on the basis of the further description herein andthe prior art on humanization referred to herein. Again, it should benoted that such humanized immunoglobulin single variable domains of theinvention can be obtained in any suitable manner known per se and thusare not strictly limited to polypeptides that have been obtained using apolypeptide that comprises a naturally occurring V_(HH) domain as astarting material.

Another particularly preferred class of immunoglobulin single variabledomains of the invention comprises immunoglobulin single variabledomains with an amino acid sequence that corresponds to the amino acidsequence of a naturally occurring V_(H) domain, but that has been“camelized”, i.e. by replacing one or more amino acid residues in theamino acid sequence of a naturally occurring V_(H) domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a V_(HH) domain of aheavy chain antibody. This can be performed in a manner known per se,which will be clear to the skilled person, for example on the basis ofthe description herein. Such “camelizing” substitutions are preferablyinserted at amino acid positions that form and/or are present at theV_(H)-V_(L) interface, and/or at the so-called Camelidae hallmark asdefined herein (see also for example WO 94/04678 and Davies andRiechmann 1994 (FEBS letters 339: 285-290) and 1996 (Protein Engineering9: 531-537)). Preferably, the V_(H) sequence that is used as a startingmaterial or starting point for generating or designing the camelizedimmunoglobulin single variable domains is preferably a V_(H) sequencefrom a mammal, more preferably the V_(H) sequence of a human being, suchas a V_(H)3 sequence. However, it should be noted that such camelizedimmunoglobulin single variable domains of the invention can be obtainedin any suitable manner known per se and thus are not strictly limited topolypeptides that have been obtained using a polypeptide that comprisesa naturally occurring V_(H) domain as a starting material.

For example, again as further described herein, both “humanization” and“camelization” can be performed by providing a nucleotide sequence thatencodes a naturally occurring V_(HH) domain or V_(H) domain,respectively, and then changing, in a manner known per se, one or morecodons in said nucleotide sequence in such a way that the new nucleotidesequence encodes a “humanized” or “camelized” immunoglobulin singlevariable domain of the invention, respectively. This nucleic acid canthen be expressed in a manner known per se, so as to provide the desiredimmunoglobulin single variable domains of the invention. Alternatively,based on the amino acid sequence of a naturally occurring V_(HH) domainor V_(H) domain, respectively, the amino acid sequence of the desiredhumanized or camelized immunoglobulin single variable domains of theinvention, respectively, can be designed and then synthesized de novousing techniques for peptide synthesis known per se. Also, based on theamino acid sequence or nucleotide sequence of a naturally occurringV_(HH) domain or V_(H) domain, respectively, a nucleotide sequenceencoding the desired humanized or camelized immunoglobulin singlevariable domains of the invention, respectively, can be designed andthen synthesized de novo using techniques for nucleic acid synthesisknown per se, after which the nucleic acid thus obtained can beexpressed in a manner known per se, so as to provide the desiredimmunoglobulin single variable domains of the invention.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said ISV is a Nanobody, a V_(HH), a humanized V_(HH), ora camelized V_(H).

Generally, proteins or polypeptides that comprise or essentially consistof a single building block, single immunoglobulin single variable domainor single Nanobody will be referred to herein as “monovalent” proteinsor polypeptides, as “monovalent constructs”, as “monovalent buildingblock”, as “monovalent immunoglobulin single variable domain”, or as“monovalent Nanobody”, respectively.

In this respect, the present invention also relates to the monovalentbuilding blocks that make up the polypeptides of the invention.

Accordingly, the present invention relates to an ISV or polypeptide thatspecifically binds the constant domain of the CD3 and that comprises oressentially consists of 4 framework regions (FR1 to FR4, respectively)and 3 complementarity determining regions (CDR1 to CDR3, respectively),in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 81-100; or        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of any of            SEQ ID NOs: 81-100, provided that the polypeptide comprising            the CDR1 with 4, 3, 2, or 1 amino acid(s) difference binds            CD3 with about the same or a higher affinity compared to the            binding by the polypeptide comprising the CDR1 without the            4, 3, 2, or 1 amino acid(s) difference, said affinity as            measured by surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 101-122; or        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of any of            SEQ ID NOs: 101-122, provided that the polypeptide            comprising the CDR2 with 4, 3, 2, or 1 amino acid(s)            difference binds CD3 with about the same or a higher            affinity compared to the binding by the polypeptide            comprising the CDR2 without the 4, 3, 2, or 1 amino acid(s)            difference, said affinity as measured by surface plasmon            resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 123-143; or        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of any of            SEQ ID NOs: 123-143, provided that the polypeptide            comprising the CDR3 with 4, 3, 2, or 1 amino acid(s)            difference binds CD3 with about the same or a higher            affinity compared to the binding by the polypeptide            comprising the CDR3 without the 4, 3, 2, or 1 amino acid(s)            difference, said affinity as measured by surface plasmon            resonance.

As discussed above, ISVs were isolated that belong to differentclusters, based on structural similarities and differences in CDR2 andCDR3.

Immunoglobulin single variable domains belonging to cluster A arerepresented by polypeptides according in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 81-87; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 81, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 101-109; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 101, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 123-127; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 123, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In another aspect, in the polypeptides belonging to cluster A, CDR1 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 81; and    -   (b) amino acid sequences that have 1 or 2 amino acid        difference(s) with SEQ ID NO: 81, wherein        -   at position 1 the G has been changed into R;        -   at position 3 the T has been changed into A;        -   at position 4 the Y has been changed into F;        -   at position 8 the S has been changed into G; and/or        -   at position 10 the G has been changed into A.

In another aspect, in the polypeptides belonging to cluster A, CDR2 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 101; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NO: 101, wherein        -   at position 3 the V has been changed into T or A;        -   at position 5 the S has been changed into T;        -   at position 6 the G has been changed into D or E; and/or        -   at position 9 the T has been changed into S, A or P.

In another aspect, in the polypeptides belonging to cluster A, CDR3 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 123; and    -   (b) amino acid sequences that have 1 or 2 amino acid        difference(s) with SEQ ID NO: 123, wherein        -   at position 2 the I has been changed into T;        -   at position 9 the I has been changed into V; and/or        -   at position 10 the A has been changed into P.

Accordingly, the present invention relates to an ISV or polypeptide thatspecifically binds CD3 and that comprises or essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

(i) CDR1 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 81; and    -   (b) amino acid sequences that have 1 or 2 amino acid        difference(s) with SEQ ID NO: 81, wherein        -   at position 1 the G has been changed into R;        -   at position 3 the T has been changed into A;        -   at position 4 the Y has been changed into F;        -   at position 8 the S has been changed into G; and/or        -   at position 10 the G has been changed into A,

and in which

(ii) CDR2 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 101; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NO: 101, wherein        -   at position 3 the V has been changed into T or A;        -   at position 5 the S has been changed into T;        -   at position 6 the G has been changed into D or E; and/or        -   at position 9 the T has been changed into S, A or P,

and in which

(iii) CDR3 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 123; and    -   (b) amino acid sequences that have 1, or 2 amino acid        difference(s) with SEQ ID NO: 123, wherein        -   at position 2 the I has been changed into T;        -   at position 9 the I has been changed into V; and/or        -   at position 10 the A has been changed into P.

In another aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR1 is represented by SEQ ID NO: 81, CDR2is represented by SEQ ID NO: 101, and CDR3 is represented by SEQ ID NO:123. Preferably the polypeptide is selected from any of SEQ ID NOs: 1 to50.

Immunoglobulin single variable domains belonging to cluster B arerepresented by polypeptides according in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 88; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 88, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 110; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 110, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 128; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 128, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In another aspect, in the polypeptides belonging to cluster B, CDR1 isSEQ ID NO: 88.

In another aspect, in the polypeptides belonging to cluster B, CDR2 isSEQ ID NO: 110.

In another aspect, in the polypeptides belonging to cluster B, CDR3 isSEQ ID NO: 128.

In another aspect, the invention relates to a polypeptide in which: CDR1is represented by SEQ ID NO: 88, CDR2 is represented by SEQ ID NO: 110,and CDR3 is represented by SEQ ID NO: 128. Preferably the polypeptide isSEQ ID NOs: 51.

Immunoglobulin single variable domains belonging to cluster C arerepresented by polypeptides according in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NO: 90; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 90, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 112-113; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 112, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NO: 130; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 130, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In another aspect, in the polypeptides belonging to cluster C, CDR1 isSEQ ID NO: 90.

In another aspect, in the polypeptides belonging to cluster C, CDR2 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 112; and    -   (b) amino acid sequence that has 1 amino acid difference with        SEQ ID NO: 112, wherein        -   at position 2 the V has been changed into A.

In another aspect, in the polypeptides belonging to cluster C, CDR3 isSEQ ID NO: 130.

Accordingly, the present invention relates to an ISV or polypeptide thatspecifically binds CD3 and that comprises or essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

(i) CDR1 is SEQ ID NO: 90; and

and in which

(ii) CDR2 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 112; and    -   (b) amino acid sequences that have 1 amino acid difference with        SEQ ID NO: 112, wherein        -   at position 2 the V has been changed into A,

and in which

(iii) CDR3 is SEQ ID NO: 130.

In another aspect, the invention relates to a polypeptide in which: CDR1is represented by SEQ ID NO: 90, CDR2 is represented by SEQ ID NO: 112,and CDR3 is represented by SEQ ID NO: 130. Preferably the polypeptide isselected from any of SEQ ID NOs: 53-56.

Immunoglobulin single variable domains belonging to cluster D arerepresented by polypeptides according in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 89; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 89, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 111; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 111, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 129; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 129, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In another aspect, in the polypeptides belonging to cluster D, CDR1 isSEQ ID NO: 89.

In another aspect, in the polypeptides belonging to cluster D, CDR2 isSEQ ID NO: 111.

In another aspect, in the polypeptides belonging to cluster D, CDR3 isSEQ ID NO: 129.

In another aspect, the invention relates to a polypeptide in which: CDR1is represented by SEQ ID NO: 89, CDR2 is represented by SEQ ID NO: 111,and CDR3 is represented by SEQ ID NO: 129. Preferably the polypeptide isSEQ ID NOs: 52.

Immunoglobulin single variable domains belonging to cluster E arerepresented by polypeptides according in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 91-93; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 91, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 114-117; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 114, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 131-133; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 131, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In another aspect, in the polypeptides belonging to cluster E, CDR1 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 91; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid(s)        difference with SEQ ID NO: 91, wherein        -   at position 6 the R has been changed into N or T;        -   at position 7 the N has been changed into H; and/or        -   at position 8 the M has been changed into T.

In another aspect, in the polypeptides belonging to cluster E, CDR2 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 114; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid(s)        difference with SEQ ID NO: 114, wherein        -   at position 1 the R has been changed into Q;        -   at position 3 the T has been changed into S; and/or        -   at position 7 the D has been changed into A or K.

In another aspect, in the polypeptides belonging to cluster E, CDR3 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 131; and    -   (b) amino acid sequences that have 1 amino acid difference with        SEQ ID NO: 131, wherein        -   at position 2 the S has been changed into R; and/or        -   at position 6 the S has been changed into V.

Accordingly, the present invention relates to an ISV or polypeptide thatspecifically binds CD3 and that comprises or essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

(i) CDR1 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 91; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NO: 91, wherein        -   at position 6 the R has been changed into N or T;        -   at position 7 the N has been changed into H; and/or        -   at position 8 the M has been changed into T,

and in which

(ii) CDR2 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 114; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NO: 114, wherein        -   at position 1 the R has been changed into Q;        -   at position 3 the T has been changed into S; and/or        -   at position 7 the D has been changed into A or K,

and in which

(iii) CDR3 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 131; and    -   (b) amino acid sequences that have 1 amino acid difference with        SEQ ID NO: 131, wherein        -   at position 2 the S has been changed into R; and/or        -   at position 6 the S has been changed into V.

In another aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR1 is represented by SEQ ID NO: 91, CDR2is represented by SEQ ID NO: 114, and CDR3 is represented by SEQ ID NO:131. Preferably the polypeptide is selected from any of SEQ ID NOs:57-65.

Immunoglobulin single variable domains belonging to cluster F arerepresented by polypeptides according in which:

-   -   (i) CDR1 is chosen from the group consisting of:        -   (a) SEQ ID NOs: 94-100; and        -   (b) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 94, provided that the polypeptide comprising the CDR1            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR1 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (ii) CDR2 is chosen from the group consisting of:        -   (c) SEQ ID NOs: 118-122; and        -   (d) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 118, provided that the polypeptide comprising the CDR2            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR2 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance; and/or    -   (iii) CDR3 is chosen from the group consisting of:        -   (e) SEQ ID NOs: 134-143; and        -   (f) amino acid sequences that have 4, 3, 2, or 1 amino            acid(s) difference with the amino acid sequence of SEQ ID            NO: 134, provided that the polypeptide comprising the CDR3            with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with            about the same or a higher affinity compared to the binding            by the polypeptide comprising the CDR3 without the 4, 3, 2,            or 1 amino acid(s) difference, said affinity as measured by            surface plasmon resonance.

In another aspect, in the polypeptides belonging to cluster F, CDR1 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 94; and    -   (b) amino acid sequences that have 1, 2, 3 or 4 amino acid(s)        difference with SEQ ID NO: 94, wherein        -   at position 3 the S has been changed into T, A or G;        -   at position 5 the N has been changed into S;        -   at position 6 the M has been changed into T or A; and/or        -   at position 9 the I has been changed into M.

In another aspect, in the polypeptides belonging to cluster F, CDR2 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 118; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid(s)        difference with SEQ ID NO: 118, wherein        -   at position 2 the H has been changed into V;        -   at position 5 the S has been changed into H or A;        -   at position 8 the N has been changed into S; and/or        -   at position 10 the Y has been changed into F.

In another aspect, in the polypeptides belonging to cluster F, CDR3 ischosen from the group consisting of

-   -   (a) SEQ ID NO: 134; and    -   (b) amino acid sequences that have 1, 2, 3, 4 or 5 amino acid(s)        difference with SEQ ID NO: 134, wherein        -   at position 6 the A has been changed into S or D;        -   at position 7 the F has been changed into Y or A;        -   at position 8 the R has been changed into H;        -   at position 9 the S has been changed into A;        -   at position 11 the G has been changed into D, T, N, S, K or            R; and/or        -   at position 14 the V has been changed into I.

Accordingly, the present invention relates to an ISV or polypeptide thatspecifically binds CD3 and that comprises or essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

(i) CDR1 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 94; and    -   (b) amino acid sequences that have 1, 2, 3 or 4 amino acid        difference(s) with SEQ ID NO: 94, wherein        -   at position 3 the S has been changed into T, A or G;        -   at position 5 the N has been changed into S;        -   at position 6 the M has been changed into T or A; and/or        -   at position 9 the L has been changed into M,

and in which

(ii) CDR2 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 118; and    -   (b) amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NO: 118, wherein        -   at position 2 the H has been changed into V;        -   at position 5 the S has been changed into H or A;        -   at position 8 the N has been changed into S; and/or        -   at position 10 the Y has been changed into F,

and in which

(iii) CDR3 is chosen from the group consisting of

-   -   (a) SEQ ID NO: 134; and    -   (b) amino acid sequences that have 1, 2, 3, 4 or 5 amino acid        difference(s) with SEQ ID NO: 134, wherein        -   at position 6 the A has been changed into S or D;        -   at position 7 the F has been changed into Y or A;        -   at position 8 the R has been changed into H;        -   at position 9 the S has been changed into A;        -   at position 11 the G has been changed into D, T, N, S, K or            R; and/or        -   at position 14 the V has been changed into I.

In another aspect, the present invention provides a polypeptide asdescribed herein, in which: CDR1 is represented by SEQ ID NO: 94, CDR2is represented by SEQ ID NO: 118, and CDR3 is represented by SEQ ID NO:134. Preferably the polypeptide is selected from any of SEQ ID NOs:66-80.

In a further aspect, the invention relates to polypeptides thatcross-block the binding to CD3 by at least one of the ISVs orpolypeptides belonging to Cluster A, B, C, D, E or F.

Accordingly, the present invention relates to polypeptides thatcross-block the binding to CD3 by at least one of the ISVs orpolypeptides with SEQ ID NOs: 1-50.

Accordingly, the present invention relates to polypeptides thatcross-block the binding to CD3 by the ISV or polypeptide with SEQ ID NO:51.

Accordingly, the present invention relates to polypeptides thatcross-block the binding to CD3 by at least one of the ISVs orpolypeptides with SEQ ID NOs: 53-56.

Accordingly, the present invention relates to polypeptides thatcross-block the binding to CD3 by the ISV or polypeptide with SEQ ID NO:52.

Accordingly, the present invention relates to polypeptides thatcross-block the binding to CD3 by at least one of the ISVs orpolypeptides with SEQ ID NOs: 57-65.

Accordingly, the present invention relates to polypeptides thatcross-block the binding to CD3 by at least one of the ISVs orpolypeptides with SEQ ID NOs: 66-80.

In a further aspect, the invention relates to polypeptides that arecross-blocked from binding to CD3 by at least one of the ISVs orpolypeptides belonging to Cluster A, B, C, D, E or F.

Accordingly, the present invention relates to polypeptides that arecross-blocked from binding to CD3 by at least one of the ISVs orpolypeptides belonging to SEQ ID NOs: 1-50.

Accordingly, the present invention relates to polypeptides that arecross-blocked from binding to CD3 by the ISV or polypeptide with SEQ IDNO: 51.

Accordingly, the present invention relates to polypeptides that arecross-blocked from binding to CD3 by at least one of the ISVs orpolypeptides belonging to SEQ ID NOs: 53-56.

Accordingly, the present invention relates to polypeptides that arecross-blocked from binding to CD3 by the ISV or polypeptide with SEQ IDNO: 52.

Accordingly, the present invention relates to polypeptides that arecross-blocked from binding to CD3 by at least one of the ISVs orpolypeptides belonging to SEQ ID NOs: 57-65.

Accordingly, the present invention relates to polypeptides that arecross-blocked from binding to CD3 by at least one of the ISVs orpolypeptides belonging to SEQ ID NOs: 66-80.

The invention further relates to compounds or constructs, and inparticular proteins or polypeptides that comprise or essentially consistof one or more ISVs or polypeptides of the invention, and optionallyfurther comprise one or more other groups, residues, moieties or bindingunits. As will become clear to the skilled person from the furtherdisclosure herein, such further groups, residues, moieties, bindingunits or amino acid sequences may or may not provide furtherfunctionality to the polypeptide of the invention (and/or to thecompound or construct in which it is present) and may or may not modifythe properties of the polypeptide of the invention.

In a specific, but non-limiting aspect of the invention, which will befurther described herein, the ISVs and polypeptides of the invention mayhave an increased half-fife in serum (as further described herein)compared to the immunoglobulin single variable domain or polypeptidefrom which they have been derived. For example, an immunoglobulin singlevariable domain or polypeptide of the invention may be linked(chemically or otherwise) to one or more groups or moieties that extendthe half-life (such as PEG), so as to provide a derivative of the ISV orpolypeptide of the invention with increased half-life.

In a specific aspect of the invention, a compound or construct of theinvention or a polypeptide of the invention may have an increasedhalf-life, compared to the corresponding ISV or polypeptide of theinvention. Some preferred, but non-limiting examples of such compounds,constructs and polypeptides will become clear to the skilled personbased on the further disclosure herein, and for example compriseimmunoglobulin single variable domains or polypeptides of the inventionthat have been chemically modified to increase the half-life thereof(for example, by means of pegylation); immunoglobulin single variabledomains or polypeptides of the invention that comprise at least oneadditional binding site for binding to a serum protein (such as serumalbumin); or constructs or polypeptides of the invention which compriseat least ISV or polypeptide of the invention that is linked to at leastone moiety (and in particular at least one amino acid sequence) whichincreases the half-life of the ISV or polypeptide of the invention.Examples of ISVs or polypeptides of the invention which comprise suchhalf-life extending moieties or immunoglobulin single variable domainswill become clear to the skilled person based on the further disclosureherein; and for example include, without limitation, polypeptides inwhich the one or more immunoglobulin single variable domains orpolypeptide of the invention are suitably linked to one or more serumproteins or fragments thereof (such as (human) serum albumin or suitablefragments thereof) or to one or more binding units that can bind toserum proteins (such as, for example, domain antibodies, immunoglobulinsingle variable domains that are suitable for use as a domain antibody,single domain antibodies, immunoglobulin single variable domains thatare suitable for use as a single domain antibody, “dAb”'s,immunoglobulin single variable domains that are suitable for use as adAb, or Nanobodies that can bind to serum proteins such as serum albumin(such as human serum albumin), serum immunoglobulins such as IgG, ortransferrin; reference is made to the further description and referencesmentioned herein); ISVs or polypeptides in which an ISV or polypeptideof the invention is linked to an Fc portion (such as a human Fc) or asuitable part or fragment thereof; or polypeptides in which the one ormore immunoglobulin single variable domains or polypeptide of theinvention are suitable linked to one or more small proteins or peptidesthat can bind to serum proteins, such as, without limitation, theproteins and peptides described in WO 91/01743, WO 01/45746, WO02/076489, WO 08/068280, WO 09/127691 and WO 11/095545.

Generally, the compounds, constructs or polypeptides of the inventionwith increased half-life preferably have a half-life that is at least1.5 times, preferably at least 2 times, such as at least 5 times, forexample at least 10 times or more than 20 times, greater than thehalf-life of the corresponding ISV or polypeptide of the invention perse. For example, the compounds, constructs or polypeptides of theinvention with increased half-life may have a half-life e.g., in humansthat is increased with more than 1 hours, preferably more than 2 hours,more preferably more than 6 hours, such as more than 12 hours, or evenmore than 24, 48 or 72 hours, compared to the corresponding ISV orpolypeptide of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchcompounds, constructs or polypeptides of the invention have a serumhalf-life e.g. in humans that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding ISV or polypeptide of the invention per se.

In another preferred, but non-limiting aspect of the invention, suchcompounds, constructs or polypeptides of the invention exhibit a serumhalf-life in human of at least about 12 hours, preferably at least 24hours, more preferably at least 48 hours, even more preferably at least72 hours or more. For example, compounds, constructs or polypeptides ofthe invention may have a half-life of at least 5 days (such as about 5to 10 days), preferably at least 9 days (such as about 9 to 14 days),more preferably at least about 10 days (such as about 10 to 15 days), orat least about 11 days (such as about 11 to 16 days), more preferably atleast about 12 days (such as about 12 to 18 days or more), or more than14 days (such as about 14 to 19 days).

In the present invention it was demonstrated that the inclusion of thealbumin targeting binding unit in the construct as such did not have anessential impact on the obtained potency or efficacy. Although a minorloss of efficacy/potency was observed in the presence of HSA, thehalf-life extended CD3 multispecific polypeptides were still potent intumour cell killing. Albumin-based drug delivery has been demonstratedto be useful for achieving improved cancer therapy, largely due to itspassive target toward tumour via the enhanced permeability and retentioneffect and the increased demand for albumin by tumour cells as source ofenergy and amino acids. However, albumin lacks not only the activemechanism to overcome the cell membrane barrier, but also the ability topenetrate into tumour tissues (Qianqian Guo et al. Polym. Chem., 2013,4, 4584-4587).

In a particularly preferred but non-limiting aspect of the invention,the invention provides a polypeptide of the invention comprising a firstand a second immunoglobulin single variable domain (ISV); and furthercomprising one or more (preferably one) serum albumin bindingimmunoglobulin single variable domain as described herein, e.g. theserum albumin binding immunoglobulin single variable domain referred toas Alb8, Alb23, Alb129, Alb132, Alb11, Alb11 (S112K)-A, Alb82, Alb82-A,Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, Alb82-GGG (Table B-3).

TABLE 8-3Immunoglobulin single variable domains for use in HLE of the ISVs andpolypeptides of the invention Alb8EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb23EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb129EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb132EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb11EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb11EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSV(S112K)-A KGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVKVSSA Alb82EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb82-AEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Alb82-AAEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAA Alb82-AAAEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAAA Alb82-GEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSG Alb82-GGEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGG Alb82-GGGEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGG

Accordingly, the present invention relates to a polypeptide as describedherein, further comprising a serum protein binding moiety.

The present invention relates to a polypeptide as described herein,wherein said serum protein binding moiety binds serum albumin.

The present invention relates to a polypeptide as described herein,wherein said serum protein binding moiety is an immunoglobulin singlevariable domain binding serum albumin.

The present invention relates to a polypeptide as described herein,wherein said ISV binding serum albumin essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively), in which CDR1 is SFGMS(SEQ ID NO: 373), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO: 374), and inwhich CDR3 is GGSLSR (SEQ ID NO: 375), CDR determined according to Kabatdefinition; and/or in which CDR1 is GFTFSSFGMS (SEQ ID NO: 376) orGFTFRSFGMS (SEQ ID NO: 377), CDR2 is SISGSGSDTL (SEQ ID NO: 378) andCDR3 is GGSLSR (SEQ ID NO: 375), CDR determined according to Kontermann2010.

The present invention relates to a polypeptide as described herein,wherein said ISV binding serum albumin comprises Alb8, Alb23, Alb129,Alb132, Alb11, Alb11 (S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA,Alb82-G, Alb82-GG, Alb82-GGG (Table B-3).

In the polypeptides of the invention, the two or more building blocks,ISVs or Nanobodies and the optionally one or more polypeptides, one ormore other groups, drugs, agents, residues, moieties or binding unitsmay be directly linked to each other (as for example described in WO99/23221) and/or may be linked to each other via one or more suitablespacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecificpolypeptides will be clear to the skilled person, and may generally beany linker or spacer used in the art to link amino acid sequences.Preferably, said linker or spacer is suitable for use in constructingproteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers thatare used in the art to link antibody fragments or antibody domains.These include the linkers mentioned in the general background art citedabove, as well as for example linkers that are used in the art toconstruct diabodies or ScFv fragments (in this respect, however, itshould be noted that, whereas in diabodies and in ScFv fragments, thelinker sequence used should have a length, a degree of flexibility andother properties that allow the pertinent V_(H) and V_(L) domains tocome together to form the complete antigen-binding site, there is noparticular limitation on the length or the flexibility of the linkerused in the polypeptide of the invention, since each ISV or Nanobody byitself forms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and inparticular amino acid sequences of between 1 and 50, preferably between1 and 30, such as between 1 and 10 amino acid residues. Some preferredexamples of such amino acid sequences include gly-ser linkers, forexample of the type (gly_(x)ser_(y))₂, such as (for example (gly₄ser)₃or (gly₃ser₂)₃, as described in WO 99/42077 and the GS30, GS15, GS9 andGS7 linkers described in the applications by Ablynx mentioned herein(see for example WO 06/040153 and WO 06/122825), as well as hinge-likeregions, such as the hinge regions of naturally occurring heavy chainantibodies or similar sequences (such as described in WO 94/04678).Preferred linkers are depicted in Table 8-4.

TABLE B-4 Linkers 5GS GGGGS 7GS SGGSGGS 9GS GGGGSGGGS 10GS GGGGSGGGGS15GS GGGGSGGGGSGGGGS 18GS GGGGSGGGGSGGGGGGGS 20GS GGGGSGGGGSGGGGSGGGGS25GS GGGGSGGGGSGGGGSGGGGSGGGGS 30GS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS Poly-A AAA

Some other particularly preferred linkers are poly-alanine (such asAAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) andGS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.For instance, polyethyleneglycol) moieties have been used to linkantibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, thedegree of flexibility and/or other properties of the linker(s) used(although not critical, as it usually is for linkers used in ScFvfragments) may have some influence on the properties of the finalpolypeptide of the invention, including but not limited to the affinity,specificity or avidity for CD3, or for one or more of the otherantigens. Based on the disclosure herein, the skilled person will beable to determine the optimal linker(s) for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

For example, in multivalent polypeptides of the invention that comprisebuilding blocks, ISVs or Nanobodies directed against a first and secondtarget, the length and flexibility of the linker are preferably suchthat it allows each building block, ISV or Nanobody of the inventionpresent in the polypeptide to bind to its cognate target, e.g. theantigenic determinant on each of the targets. Again, based on thedisclosure herein, the skilled person will be able to determine theoptimal linker(s) for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

It is also within the scope of the invention that the linker(s) usedconfer one or more other favourable properties or functionality to thepolypeptides of the invention, and/or provide one or more sites for theformation of derivatives and/or for the attachment of functional groups(e.g. as described herein for the derivatives of the ISV, Nanobodies, orpolypeptide of the invention). For example, linkers containing one ormore charged amino acid residues can provide improved hydrophilicproperties, whereas linkers that form or contain small epitopes or tagscan be used for the purposes of detection, Identification and/orpurification. Again, based on the disclosure herein, the skilled personwill be able to determine the optimal linkers for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

Finally, when two or more linkers are used in the polypeptides of theinvention, these linkers may be the same or different. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of theinvention will be a linear polypeptide. However, the invention in itsbroadest sense is not limited thereto. For example, when a polypeptideof the invention comprises three of more building blocks, ISV orNanobodies, it is possible to link them by use of a linker with three ormore “arms”, which each “arm” being linked to a building block, ISV orNanobody, so as to provide a “star-shaped” construct. It is alsopossible, although usually less preferred, to use circular constructs.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said first ISV and said second ISV and possibly saidthird ISV and/or said ISV binding serum albumin are directly linked toeach other or are linked via a linker.

The present invention relates to a polypeptide as described herein,wherein said linker is chosen from the group consisting of linkers of5GS, 7G5, 9G5, 10GS, 15GS, 18G5, 20GS, 25GS, 30GS and 35GS.

The present invention relates to a polypeptide as described herein,wherein said serum protein binding moiety is a non-antibody basedpolypeptide (e.g. PEG).

The invention also relates to methods for preparing the ISVs,polypeptides and constructs described herein. The ISVs, polypeptides andconstructs of the invention can be prepared in a manner known per se, aswill be clear to the skilled person from the further description herein.For example, the ISVs, polypeptides and constructs of the invention canbe prepared in any manner known per se for the preparation of antibodiesand in particular for the preparation of antibody fragments (includingbut not limited to (single) domain antibodies and ScFv fragments). Somepreferred, but non-limiting methods for preparing the polypeptides andconstructs include the methods and techniques described herein.

The method for producing an ISV, polypeptide or protein construct of theinvention may comprise the following steps:

-   -   the expression, in a suitable host cell or host organism (also        referred to herein as a “host of the invention”) or in another        suitable expression system of a nucleic acid that encodes said        IVS, polypeptide or protein construct of the invention,    -   optionally followed by:    -   isolating and/or purifying the ISVs, polypeptide or protein        construct of the invention thus obtained.

In particular, such a method may comprise the steps of:

-   -   cultivating and/or maintaining a host of the invention under        conditions that are such that said host of the invention        expresses and/or produces at least one ISVs, polypeptide or        protein construct of the invention;    -   optionally followed by:    -   isolating and/or purifying the ISVs, polypeptide or protein        construct of the invention thus obtained.

Accordingly, the present invention also relates to a nucleic acid ornucleotide sequence that encodes an ISV, polypeptide or proteinconstruct of the invention (also referred to as “nucleic acid of theinvention” or “nucleotide sequence of the invention”). A nucleic acid ofthe invention can be in the form of single or double stranded DNA orRNA, and is preferably in the form of double stranded DNA. For example,the nucleotide sequences of the invention may be genomic DNA, cDNA orsynthetic DNA (such as DNA with a codon usage that has been specificallyadapted for expression in the intended host cell or host organism).

According to one embodiment of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined herein. Thenucleic acid of the invention may also be in the form of, be present inand/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the polypeptides orprotein constructs of the invention given herein, and/or can be isolatedfrom a suitable natural source. Also, as will be clear to the skilledperson, to prepare a nucleic acid of the invention, also severalnucleotide sequences, such as at least one nucleotide sequence encodingan immunoglobulin single variable domain of the invention and forexample nucleic acids encoding one or more linkers can be linkedtogether in a suitable manner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers. These and other techniques will beclear to the skilled person, and reference is again made to the standardhandbooks, such as Sambrook et al. and Ausubel et al., mentioned herein,as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art. Such genetic constructs generally comprise at leastone nucleic acid of the invention that is optionally linked to one ormore elements of genetic constructs known per se, such as for exampleone or more suitable regulatory elements (such as a suitablepromoter(s), enhancer(s), terminator(s), etc.) and the further elementsof genetic constructs referred to herein. Such genetic constructscomprising at least one nucleic acid of the invention will also bereferred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable forindependent replication, maintenance and/or inheritance in the intendedhost organism. For instance, the genetic constructs of the invention maybe in the form of a vector, such as for example a plasmid, cosmid, YAC,a viral vector or transposon. In particular, the vector may be anexpression vector, i.e. a vector that can provide for expression invitro and/or in vivo (e.g. in a suitable host cell, host organism and/orexpression system).

In a preferred but non-limiting embodiment, a genetic construct of theinvention comprises

-   -   a) at least one nucleic acid of the invention; operably        connected to    -   b) one or more regulatory elements, such as a promoter and        optionally a suitable terminator;

and optionally also

-   -   c) one or more further elements of genetic constructs known per        se;

in which the terms “regulatory element”, “promoter”, “terminator” and“operably connected” have their usual meaning in the art (as furtherdescribed herein); and in which said “further elements” present in thegenetic constructs may for example be 3′- or 5′-UTR sequences, leadersequences, selection markers, expression markers/reporter genes, and/orelements that may facilitate or increase (the efficiency of)transformation or integration. These and other suitable elements forsuch genetic constructs will be clear to the skilled person, and may forinstance depend upon the type of construct used; the intended host cellor host organism; the manner in which the nucleotide sequences of theinvention of interest are to be expressed (e.g. via constitutive,transient or inducible expression); and/or the transformation techniqueto be used. For example, regulatory sequences, promoters and terminatorsknown per se for the expression and production of antibodies andantibody fragments (including but not limited to (single) domainantibodies and ScFv fragments) may be used in an essentially analogousmanner.

Preferably, in the genetic constructs of the invention, said at leastone nucleic acid of the invention and said regulatory elements, andoptionally said one or more further elements, are “operably linked” toeach other, by which is generally meant that they are in a functionalrelationship with each other. For instance, a promoter is considered“operably linked” to a coding sequence if said promoter is able toinitiate or otherwise control/regulate the transcription and/or theexpression of a coding sequence (in which said coding sequence should beunderstood as being “under the control of” said promoter). Generally,when two nucleotide sequences are operably linked, they will be in thesame orientation and usually also in the same reading frame. They willusually also be essentially contiguous, although this may also not berequired.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.for expression and/or production of the polypeptide or protein constructof the invention. The host is preferably a non-human host. Suitablehosts or host cells will be clear to the skilled person, and may forexample be any suitable fungal, prokaryotic or eukaryotic cell or cellline or any suitable fungal, prokaryotic or eukaryotic organism, forexample:

-   -   a bacterial strain, including but not limited to gram-negative        strains such as strains of Escherichia coli; of Proteus, for        example of Proteus mirabilis; of Pseudomonas, for example of        Pseudomonas fluorescens; and gram-positive strains such as        strains of Bacillus, for example of Bacillus subtilis or of        Bacillus brevis; of Streptomyces, for example of Streptomyces        lividans; of Staphylococcus, for example of Staphylococcus        carnosus; and of Lactococcus, for example of Lactococcus lactis;    -   a fungal cell, including but not limited to cells from species        of Trichoderma, for example from Trichoderma reesei; of        Neurospora, for example from Neurospora crassa; of Sordaria, for        example from Sordaria macrospora; of Aspergillus, for example        from Aspergillus niger or from Aspergillus sojae; or from other        filamentous fungi;    -   a yeast cell, including but not limited to cells from species of        Saccharomyces, for example of Saccharomyces cerevisiae; of        Schizosaccharomyces, for example of Schizosaccharomyces pombe;        of Pichia, for example of Pichia pastoris or of Pichia        methanolica; of Hansenula, for example of Hansenula polymorphs;        of Kluyveromyces, for example of Kluyveromyces lactis; of        Arxula, for example of Arxula adeninivorans; of Yarrowia, for        example of Yarrowia lipolytica;    -   an amphibian cell or cell line, such as Xenopus oocytes;    -   an insect-derived cell or cell line, such as cells/cell lines        derived from lepidoptera, including but not limited to        Spodoptera SF9 and Sf21 cells or cells/cell lines derived from        Drosophila, such as Schneider and Kc cells;    -   a plant or plant cell, for example in tobacco plants; and/or    -   a mammalian cell or cell line, for example a cell or cell line        derived from a human, a cell or a cell line from mammals        including but not limited to CHO-cells, BHK-cells (for example        BHK-21 cells) and human cells or cell lines such as HeLa, COS        (for example COS-7) and PER.C6 cells;

as well as all other hosts or host cells known per se for the expressionand production of antibodies and antibody fragments (including but notlimited to (single) domain antibodies and ScFv fragments), which will beclear to the skilled person. Reference is also made to the generalbackground art cited hereinabove, as well as to for example WO 94/29457;WO 96/34103; WO 99/42077; Frenken et al. 1998 (Res. Immunol. 149:589-99); Riechmann and Muyldermans 1999 (J. Immunol. Met. 231: 25-38);van der Linden 2000 (J. Biotechnol. 80: 261-70); Joosten et al. 2003(Microb. Cell Fact. 2: 1); Joosten et al. 2005 (Appl. Microbiol.Biotechnol. 66: 384-92); and the further references cited herein.

For expression of the ISVs, polypeptides or constructs in a cell, theymay also be expressed as so-called “intrabodies”, as for exampledescribed in WO 94/02610, WO 95/22618 and U.S. Pat. No. 7,004,940; WO03/014960; in Cattaneo and Biocca 1997 (Intracellular Antibodies:Development and Applications. Landes and Springer-Verlag); and inKontermann 2004 (Methods 34: 163-170).

According to one preferred, but non-limiting embodiment of theinvention, the ISV, polypeptide or protein construct of the invention isproduced in a bacterial cell, in particular a bacterial cell suitablefor large scale pharmaceutical production, such as cells of the strainsmentioned above.

According to another preferred, but non-limiting embodiment of theinvention, the ISV, polypeptide or protein construct of the invention isproduced in a yeast cell, in particular a yeast cell suitable for largescale pharmaceutical production, such as cells of the species mentionedabove.

According to yet another preferred, but non-limiting embodiment of theinvention, the ISV, polypeptide or construct of the invention isproduced in a mammalian cell, in particular in a human cell or in a cellof a human cell line, and more in particular in a human cell or in acell of a human cell line that is suitable for large scalepharmaceutical production, such as the cell lines mentioned hereinabove.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above.

After transformation, a step for detecting and selecting those hostcells or host organisms that have been successfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct of the invention or a step involvingthe detection of the polypeptide of the invention, e.g. using specificantibodies.

The transformed host cell (which may be in the form or a stable cellline) or host organisms (which may be in the form of a stable mutantline or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that theyexpress, or are (at least) capable of expressing (e.g. under suitableconditions), an ISV, polypeptide or protein construct of the invention(and in case of a host organism: in at least one cell, part, tissue ororgan thereof). The invention also includes further generations, progenyand/or offspring of the host cell or host organism of the invention, forinstance obtained by cell division or by sexual or asexual reproduction.

Accordingly, in another aspect, the invention relates to a host or hostcell that expresses (or that under suitable circumstances is capable ofexpressing) an ISV, polypeptide or protein construct of the invention;and/or that contains a nucleic acid encoding the same. Some preferredbut non-limiting examples of such hosts or host cells can be asgenerally described in WO 04/041867, WO 04/041865 or WO 09/068627. Forexample, ISVs, polypeptides and protein constructs of the invention maywith advantage be expressed, produced or manufactured in a yeast strain,such as a strain of Pichia pastoris. Reference is also made to WO04/25591, WO 10/125187, WO 11/003622, and WO 12/056000 which alsodescribes the expression/production in Pichia and other hosts/host cellsof immunoglobulin single variable domains and polypeptides comprisingthe same.

To produce/obtain expression of the ISVs, polypeptides or proteinconstructs of the invention, the transformed host cell or transformedhost organism may generally be kept, maintained and/or cultured underconditions such that the (desired) ISV, polypeptide or protein constructof the invention is expressed/produced. Suitable conditions will beclear to the skilled person and will usually depend upon the hostcell/host organism used, as well as on the regulatory elements thatcontrol the expression of the (relevant) nucleotide sequence of theinvention. Again, reference is made to the handbooks and patentapplications mentioned above in the paragraphs on the genetic constructsof the invention.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); ail of whichmay be selected by the skilled person. Again, under such conditions, theISVs, polypeptides or protein constructs of the invention may beexpressed in a constitutive manner, in a transient manner, or only whensuitably induced.

It will also be clear to the skilled person that the ISV, polypeptide orprotein construct of the invention may (first) be generated in animmature form (as mentioned above), which may then be subjected topost-translational modification, depending on the host cell/hostorganism used. Also, the ISV, polypeptide or protein construct of theinvention may be glycosylated, again depending on the host cell/hostorganism used.

The ISV, polypeptide or protein construct of the invention may then beisolated from the host cell/host organism and/or from the medium inwhich said host cell or host organism was cultivated, using proteinisolation and/or purification techniques known per se, such as(preparative) chromatography and/or electrophoresis techniques,differential precipitation techniques, affinity techniques (e.g. using aspecific, cleavable amino acid sequence fused with the polypeptide orconstruct of the invention) and/or preparative immunological techniques(i.e. using antibodies against the amino acid sequence to be isolated).

The constructs of the invention can generally be prepared by a methodwhich comprises at least the step of suitably linking ISVs orpolypeptides of the invention to the one or more further groups,residues, moieties or binding units, optionally via the one or moresuitable linkers, so as to provide the constructs of the invention. TheISVs, polypeptides and constructs of the invention can then further bemodified, and in particular by chemical and/or biological (e.g.enzymatical) modification, of one or more of the amino acid residuesthat form the polypeptides or constructs of the invention, to obtainderivatives of the polypeptides or constructs of the invention.

The invention also relates to a pharmaceutical composition comprisingthe ISV, polypeptide, compound or construct of the invention.

In the above methods, the amino acid sequences, ISVs, Nanobodies,polypeptides, compounds or constructs of the invention and/or thecompositions comprising the same can be administered in any suitablemanner, depending on the specific pharmaceutical formulation orcomposition to be used.

Thus, the amino acid sequences, ISVs, Nanobodies, polypeptides,compounds or constructs of the invention and/or the compositionscomprising the same can for example be administered orally,intraperitoneally (e.g. intravenously, subcutaneously, intramuscularly,or via any other route of administration that circumvents thegastrointestinal tract), intranasally, transdermally, topically, bymeans of a suppository, by inhalation, again depending on the specificpharmaceutical formulation or composition to be used. The clinician willbe able to select a suitable route of administration and a suitablepharmaceutical formulation or composition to be used in suchadministration, depending on the disease or disorder to be prevented ortreated and other factors well known to the clinician.

As used herein, the term “therapeutic agent” refers to any agent thatcan be used in the treatment and/or management of a hyperproliferativecell disorder, e.g., cancer, or one or more symptoms thereof. In certainembodiments, the term “therapeutic agent” refers to a multispecificpolypeptide of the invention. Preferably, a therapeutic agent is anagent which is known to be useful for, or has been or is currently beingused for the treatment, prevention and/or management of ahyperproliferative cell disorder disorder, e.g., cancer, or one or moresymptoms thereof.

As used herein, a “therapeutically effective amount” in the context ofcancer refers to the amount of a therapy alone, or in combination withother therapies, that provides a therapeutic benefit in the treatmentand/or management of cancers. In one aspect, a therapeutically effectiveamount refers to the amount of a therapy sufficient to destroy, modify,control or remove primary, regional or metastatic cancer tissue. Inanother aspect, a therapeutically effective amount refers to the amountof a therapy sufficient to reduce the symptoms of a cancer. In anotheraspect, a therapeutically effective amount refers to the amount of atherapy sufficient to delay or minimize the spread of cancer. In aspecific embodiment, a therapeutically effective amount of a therapy isan amount of a therapy sufficient to inhibit growth or proliferation ofcancer cells, kill existing cancer cells (e.g., cause regression of thecancer), and/or prevent the spread of cancer cells to other tissues orareas (e.g., prevent metastasis). In another specific embodiment, atherapeutically effective amount of a therapy is the amount of a therapysufficient to inhibit the growth of a tumor by at least 5%, preferablyat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 100% as measured by astandard method known in the art. Used in connection with an amount of amultispecific polypeptide of the invention, the term can encompass anamount that improves overall therapy, reduces or avoids unwantedeffects, or enhances the therapeutic efficacy of or synergies withanother therapy. In one embodiment, a therapeutically effective amountof a therapy reduces or avoids unwanted effects, or enhances thetherapeutic efficacy of or synergies with another therapy by at least5%, preferably at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 100%relative to a control (e.g., a negative control such as phosphatebuffered saline) in an assay known in the art or described herein.

As used herein, a “therapeutically effective amount” in the context of anon-cancer hyperproliferative cell disorder refers to the amount of atherapy alone, or in combination with other therapies, that provides atherapeutic benefit in the treatment and/or management of said disorder.In one aspect, a therapeutically effective amount refers to the amountof a therapy sufficient to destroy, modify, control or remove cellsaffected by a non-cancer hyperproliferative cell disorder. In anotheraspect, a therapeutically effective amount refers to the amount of atherapy sufficient to reduce the symptoms of a non-cancerhyperproliferative cell disorder. In another aspect, a therapeuticallyeffective amount refers to the amount of a therapy sufficient to delayor minimize the spread of the non-cancer hyperproliferative celldisorder. In a specific embodiment, a therapeutically effective amountof a therapy is an amount of a therapy sufficient to inhibit growth orproliferation of the non-cancer hyperproliferative cell disorder, killexisting non-cancer hyperproliferative cells (e.g., cause regression ofthe disorder). In another specific embodiment, a therapeuticallyeffective amount of a therapy is the amount of a therapy sufficient toinhibit the growth of the non-cancer hyperproliferative cells by atleast 5%, preferably at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least100% as measured by a standard method known in the art. Used inconnection with an amount of a multispecific polypeptide of theinvention, the term can encompass an amount that improves overalltherapy, reduces or avoids unwanted effects, or enhances the therapeuticefficacy of or synergies with another therapy. In one embodiment, atherapeutically effective amount of a therapy reduces or avoids unwantedeffects, or enhances the therapeutic efficacy of or synergies withanother therapy by at least 5%, preferably at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 100% relative to a control (e.g., a negativecontrol such as phosphate buffered saline) in an assay known in the art.

As used herein, the term “therapy” refers to any protocol, method and/oragent that can be used in the treatment, prevention and/or management ofa hyperproliferative cell disorder, e.g., cancer. In certainembodiments, the terms “therapies” and “therapy” refer to a biologicaltherapy, supportive therapy, and/or other therapies useful in thetreatment, prevention and/or management of a hyperproliferative celldisorder, e.g., cancer, or one or more symptoms thereof known to one ofskill in the art such as medical personnel.

As used herein, the terms “treat”, “treatment” and “treating” in thecontext of administering a therapy(ies) to a subject refer to thereduction or amelioration of the progression, severity, and/or durationof a disorder associated with a hyperproliferative cell disorder, e.g.,cancer, and/or the amelioration of one or more symptoms thereofresulting from the administration of one or more therapies (including,but not limited to, the administration of one or more prophylactic ortherapeutic agents). In specific embodiments, the terms “treat”,“treatment” and “treating” in the context of administering (a)therapy(ies) to a subject refer to the reduction or amelioration of theprogression, severity, and/or duration of a hyperproliferative celldisorder, e.g., cancer, refers to a reduction in cancer cells by atleast 5%, preferably at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least100% relative to a control (e.g., a negative control such as phosphatebuffered saline). In other embodiments, the terms “treat”, “treatment”and “treating” in the context of administering (a) therapy(ies) to asubject refer to the reduction or amelioration of the progression,severity, and/or duration of a hyperproliferative cell disorder, e.g.,cancer, refers to no change in cancer cell number, a reduction inhospitalization time, a reduction in mortality, or an increase insurvival time of the subject with cancer.

The amino acid sequences, ISVs, Nanobodies, polypeptides, compoundsand/or constructs of the invention and/or the compositions comprisingthe same are administered according to a regime of treatment that issuitable for preventing and/or treating the hyperproliferative celldisorder, e.g., cancer, to be prevented or treated. The clinician willgenerally be able to determine a suitable treatment regimen, dependingon factors such as the stage of the hyperproliferative cell disorder,e.g., cancer, to be treated, the severity of the hyperproliferative celldisorder, e.g., cancer, to be treated and/or the severity of thesymptoms thereof, the specific amino acid sequence, ISV, Nanobody,polypeptide, compound and/or construct of the invention to be used, thespecific route of administration and pharmaceutical formulation orcomposition to be used, the age, gender, weight, diet, general conditionof the patient, and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more amino acid sequences, ISVs, Nanobodies, polypeptides, compoundsand/or constructs of the invention, or of one or more compositionscomprising the same, in one or more pharmaceutically effective amountsor doses. The specific amount(s) or doses to be administered can bedetermined by the clinician, again based on the factors cited above.

Generally, for the prevention and/or treatment of a hyperproliferativecell disorder, e.g., cancer, mentioned herein and depending on the typeof hyperproliferative cell disorder, e.g., cancer, and stage of thedisease to be treated, the potency of the specific amino acid sequence,ISV, Nanobody, polypeptide, compound or construct of the invention to beused, the specific route of administration and the specificpharmaceutical formulation or composition used, the amino acidsequences, ISVs, Nanobodies, polypeptides, compounds or constructs ofthe invention will generally be administered in an amount between 1 gramand 0.01 milligram per kg body weight per day, preferably between 0.1gram and 0.01 milligram per kg body weight per day, such as about 0.1,1, 10, 100 or 1000 milligram per kg body weight per day, e.g. from 0.1mg per kg to 25 mg per kg of the subject's body weight; eithercontinuously (e.g. by infusion), as a single daily dose or as multipledivided doses during the day. The clinician will generally be able todetermine a suitable daily dose, depending on the factors mentionedherein. It will also be clear that in specific cases, the clinician maychoose to deviate from these amounts, for example on the basis of thefactors cited above and his expert judgment. Generally, some guidance onthe amounts to be administered can be obtained from the amounts usuallyadministered for comparable conventional antibodies or antibodyfragments against the same target administered via essentially the sameroute, taking into account however differences in affinity/avidity,efficacy, biodistribution, half-life and similar factors well known tothe skilled person.

Usually, in the above method, a single amino acid sequence, ISV,Nanobody, polypeptide, compound or construct of the invention will beused. It is however within the scope of the invention to use two or moreamino acid sequences, ISVs, Nanobodies, polypeptides compounds and/orconstructs of the invention in combination.

The ISVs, Nanobodies, amino acid sequences, polypeptides, compoundsand/or constructs of the invention may also be used in combination withone or more further pharmaceutically active compounds or principles,i.e. as a combined treatment regimen, which may or may not lead to asynergistic effect. Again, the clinician will be able to select suchfurther compounds or principles, as well as a suitable combinedtreatment regimen, based on the factors cited above and his expertjudgement.

In particular, the amino acid sequences, ISVs, Nanobodies, polypeptides,compounds and/or constructs of the invention may be used in combinationwith other pharmaceutically active compounds or principles that are orcan be used for the prevention and/or treatment of thehyperproliferative cell disorder, e.g., cancer, disease and/or disordercited herein, as a result of which a synergistic effect may or may notbe obtained. Examples of such compounds and principles, as well asroutes, methods and pharmaceutical formulations or compositions foradministering them will be clear to the clinician.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

In one aspect, the disclosure provides methods for the administration ofimmunoglobulin single variable domains and polypeptide constructsthereof comprising one or more immunoglobulin single variable domains,polypeptides, compounds and/or constructs. In some embodiments, theimmunoglobulin single variable domain, polypeptide, compound and/orconstruct is administered as a pharmaceutical composition. Thepharmaceutical composition, in addition to the immunoglobulin singlevariable domains and polypeptide constructs thereof includes apharmaceutically-acceptable carrier.

As described in detail, the pharmaceutical compositions of thedisclosure may be specially formulated for administration in solid orliquid form, including those adapted for the following: oraladministration, for example, drenches (aqueous or non-aqueous solutionsor suspensions), tablets, e.g., those targeted for buccal, sublingual,and systemic absorption, boluses, powders, granules, pastes forapplication to the tongue; parenteral administration, for example, bysubcutaneous, intramuscular, intravenous or epidural injection as, forexample, a sterile solution or suspension, or sustained-releaseformulation; topical application, for example, as a cream, ointment, ora controlled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream or foam; sublingually; ocularly; transdermally; or nasally,pulmonary and to other mucosal surfaces.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

Formulations of the disclosure include those suitable for oral, nasal,topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient (e.g.,immunoglobulin single variable domain or polypeptide constructs thereof)which can be combined with a carrier material to produce a single dosageform will vary depending upon the host being treated, and the particularmode of administration. The amount of active ingredient that can becombined with a carrier material to produce a single dosage form willgenerally be that amount of the compound which produces a therapeuticeffect. Generally, this amount will range from about 1% to about 99% ofactive ingredient, preferably from about 5% to about 70%, mostpreferably from about 10% to about 30%.

In certain embodiments, a formulation comprises an excipient selectedfrom the group consisting of cyclodextrins, liposomes, micelle formingagents, e.g., bile acids, and polymeric carriers, e.g., polyesters andpolyanhydrides. In certain embodiments, an aforementioned formulationrenders orally bioavailable an immunoglobulin single variable domain orpolypeptide construct.

Methods of preparing these formulations or compositions include the stepof bringing into association an immunoglobulin single variable domain orpolypeptide construct with the carrier and, optionally, one or moreaccessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing into association an immunoglobulinsingle variable domain or polypeptide construct with liquid carriers, orfinely divided solid carriers, or both, and then, if necessary, shapingthe product.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of an immunoglobulin single variable domain orpolypeptide construct as an active ingredient. An immunoglobulin singlevariable domain or polypeptide construct invention may also beadministered as a bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically-acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia;humectants, such as glycerol; disintegrating agents, such as agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certainsilicates, and sodium carbonate; solution retarding agents, such asparaffin; absorption accelerators, such as quaternary ammoniumcompounds; wetting agents, such as, for example, cetyl alcohol, glycerolmonostearate, and non-ionic surfactants; absorbents, such as kaolin andbentonite clay; lubricants, such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxy-propylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made in asuitable machine in which a mixture of the powdered compound ismoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions, such as dragees, capsules, pills and granules, mayoptionally be scored or prepared with coatings and shells, such asenteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions that can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active ingredient, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions for rectal or vaginaladministration may be presented as a suppository, which may be preparedby mixing an immunoglobulin single variable domain or polypeptideconstruct with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations suitable for vaginal administration also include pessaries,tampons, creams, gels, pastes, foams or spray formulations containingsuch carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of animmunoglobulin single variable domain or polypeptide construct includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically-acceptable carrier, and with anypreservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, excipients, such asanimal and vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamide powder, ormixtures of these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of an immunoglobulin single variable domain or polypeptideconstruct to the body. Dissolving or dispersing the compound in theproper medium can make such dosage forms. Absorption enhancers can alsobe used to increase the flux of the compound across the skin. Eitherproviding a rate controlling membrane or dispersing the compound in apolymer matrix or gel can control the rate of such flux.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this disclosure.

Pharmaceutical compositions suitable for parenteral administrationcomprise one or more an immunoglobulin single variable domains orpolypeptide constructs in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain sugars, alcohols,antioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers, which may beemployed in the pharmaceutical compositions include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Properfluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly-(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissue.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Modifications and variationof the above-described embodiments of the invention are possible withoutdeparting from the invention, as appreciated by those skilled in the artin light of the above teachings. It is therefore understood that, withinthe scope of the claims and their equivalents, the invention may bepracticed otherwise than as specifically described.

The invention will now be further described by means of the followingnon-limiting preferred aspects, examples and figures.

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference, in particular for theteaching that is referenced hereinabove.

EXAMPLES Example 1 TCR αβ/CD3 Cell Lines

Transient and stable CHO-K1 (ATCC: CCL-61), HEK293H (Life technologies11631-017), Llana (Fibroblast cells from llama Navel cord cells) celllines with recombinant overexpression of all 6 chains of the full humanT-cell Receptor complex were generated. For this, the coding sequencesof the TCR alpha (α) and TCR beta (β) chain were cloned in apcDNA3.1-derived vector, downstream of a CMV promotor and a 2A-likeviral peptide sequence was inserted between both chains to induceribosomal skipping during translation of the polyprotein. In the samevector, the coding sequences of the epsilon, delta, gamma and zetachains of the CD3 complex were cloned downstream of an additional CMVpromotor, also using 2A-like viral peptide sequences between therespective chains. In addition, a stable HEK293H clone with recombinantoverexpression of the 4 chains of the human CD3 was generated asdescribed above using a single gene vector.

The sequences for the human CD3 and the human TCRα/β constant domainswere retrieved from UniProtKB (CD3 delta: P04234, CD3 gamma: P09693, CD3epsilon: P07766, CD3 zeta: P20963, TCRα: P01848 and TCRβ: P01850). Thesequences for the human TCRα/β variable domains were retrieved fromcrystal structure. (PDB codes: 2IAN, 2XN9 and 3TOE).

The cell surface expression of the human T cell receptor complex wasconfirmed using a functional mouse IgG2b anti-human TCRα/β antibody,clone BW242/412 (Miltenyi 130-098-219) and a functional mouse IgG2aanti-CD3 PE labelled antibody, clone OKT-3 (eBioscience 12-0037). (FIG.1)

Example 2 Immunization of Llamas with TCR/CD3, Cloning of the HeavyChain-Only Antibody Fragment Repertoires and Preparation of Phage

2.1 Immunization

After approval of the Ethical Committee (Faculty of Veterinary Medicineof the University of Ghent, Belgium-EC2004/044 en EC2005/053), Llamas(Ilama glama) were immunized with either human peripheral bloodlymphocytes (PBMC) isolated by Ficol density centrifugation from asingle buffy coat, or with mouse or human T- and NK cells enriched fromPBMC by magnetic cell separation using biotin-conjugated antibodies.None of these immunization strategies resulted in specific immuneresponse.

After approval of the Ethical Committee (CRIA, LA1400575,Belgium-EC2012#1), 3 additional llamas were immunized with a pVAX1-humanTCR(2IAN)/CD3 (described in Example 1) plasmid vector (Invitrogen,Carlsbad, Calif., USA) and with a pVAX1-human TCR(2XN9)/CD3 (describedin Example 1) plasmid vector (Invitrogen, Carlsbad, Calif., USA)according to standard protocols. Two llamas received additionally 1subcutaneous injection of primary human T cells. Human T Cells werecollected from Buffy Coat blood, from healthy volunteers (Blood bankGent) using RosetteSep (StemCell Technologies, #15061) followed by anenrichment on Ficoll-Paque™ PLUS (GE Healthcare #17-1440-03) accordingto manufactures instructions and stored in liquid nitrogen. Afterthawing, cells were washed, and re-suspended in D-PBS from Gibco andkept on ice prior to injection.

2.2 Cloning of the Heavy Chain-Only Antibody Fragment Repertoires andPreparation of Phages.

Per animal, blood samples were collected after the injection of one typeof immunization antigen. From these blood samples, PBMC were preparedusing Ficoll-Hypaque according to the manufacturer's instructions(Amersham Biosciences, Piscataway, N.J., USA). For each immunized llama,libraries were constructed by pooling the total RNA isolated fromsamples originating from a certain subset of the immunization schedule,i.e. after one type of immunization antigen.

In short, the PCR-amplified VHH repertoire was cloned via specificrestriction sites into a vector designed to facilitate phage display ofthe VHH library. The vector was derived from pUC119. In frame with theVHH coding sequence, the vector encodes a C-terminal 3×FLAG and His6tag. Phages were prepared according to standard protocols (see forexample WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858 and otherprior art and applications filed by Ablynx N.V. cited herein).

Example 3 Selection of TCR/CD3 Specific VHHs Via Phage Display

VHH repertoires obtained from all llamas and cloned as phage librarywere used in different selection strategies, applying a multiplicity ofselection conditions. Variables included: 1) the presentation form ofthe human TCR α/β/CD3 (on different cell backgrounds or on purifiedprimary T cells (isolated as described in Example 2.1, ii) the antigenconcentration, iii) the number of selection rounds. In brief cells wereincubated for 2 h with the phage libraries followed by extensivewashing; bound phages were eluted with trypsin (1 mg/mL) for 15 minutes.When trypsin was used for phage elution, the protease activity wasimmediately neutralized by applying 0.8 mM protease inhibitor ABSF. Ascontrol, selections with parental cell line or without antigen wereperformed in parallel.

Phage outputs were used to infect E. coli for analysis of individual VHHclones. Periplasmic extracts were prepared according to standardprotocols (see for example WO 03/035694, WO 04/041865, WO 04/041863, WO04/062551 and other prior art and applications filed by Ablynx N.V.cited herein).

Example 4 Screening

4.1 Screening for TCR/CD3 Binding Nanobodies in a Flow Cytometry Assay

Periplasmic extracts were screened for cell expressed TCR/CD3 bindingusing human TCR/CD3 transfected CHO-K1 or HEK293H cells and therespective CHO-K1 or HEK293H reference cell line in a mixed cell linesetup. To this end, a large batch of the reference cell lines werelabelled with 8 μM PKH26 and frozen. 5×10⁴ PKH labelled reference cellswere mixed with 5×10⁴ target cells and incubated with periplasmicextracts for 30 min at 4° C., and washed 3 times. Next, cells wereincubated with 1 μg/ml monoclonal ANTI-FLAG® M2 antibody (Sigma-Aldrich,cat# F1804) for 30 min at 4° C., washed again, and incubated for 30 minat 4° C. with goat anti-mouse APC labelled antibody (JacksonImmunoresearch 115-135-164, 1:100). Samples were washed, resuspended inFACS Buffer (D-PBS from Gibco, with 10% FBS from Sigma and 0.05% sodiumazide from Merck) and then analysed on a BD FACSArray. First a P1population which represents more than 80% of the total cell populationwas selected based on FSC-SSC distribution. In this gate, 20,000 cellswere counted during acquisition. Based on PKI-126-SSC distribution, thePKH labelled parental population and the human TCR/CD3 unlabelled targetpopulation was selected. For these 2 populations the mean APC value wascalculated.

4.2 Screening for TCR/CD3 Binding Nanobodies in a Human T CellActivation Assay

After several attempts, it turned out that activation of purified humanT cells by antibodies or Nanobodies according to standard protocols,i.e. coated onto a 96 well plate, was not sensitive enough (data notshown).

In order to assess activity, a different assay was developed, based on abead coupled T cell activation. In short, goat anti-mouse IgG dynabeads(Life technologies #11033) were coated with mouse anti-flag IgGantibodies (Sigma F1804), (15 μg/1E7 beads). After an incubation periodof 2 h at 4° C., beads were washed and incubated with 80111 periplasmicextract for 20 min at 4° C. while shaking. Non-coupled Nanobodies werewashed away before adding the bead complex together with soluble mouseanti-CD28 antibody (Pelicluster CD28—Sanquin #M1650) to purified primaryhuman T cells (isolated as described in Example 2.1). As controlcondition, non-stimulated human T cells were used. In brief, goatanti-mouse IgG dynabeads coupled to mouse anti-flag IgG were incubatedin 80 μl periplasmic extract containing irrelevant Nanobodies. Afterremoval of the non-coupled Nanobodies during a wash step the irrelevantNanobody-bead complex was added to purified primary human T cells.

After an incubation of 24 h at 37° and 5% CO₂ the activation status ofthe human T cells was determined by measuring the CD69 expression levelin flow cytometry using monoclonal mouse anti-human CD69PE (BD #557050).

Nanobodies which scored positive in the flow cytometric binding screenand the T cell activation assay were sequenced.

The sequence analysis resulted in the identification of 6 distinctclusters. Corresponding alignments are provided (Table A-1, Table A-2,Table A-3, Table A-4, Table A-5, Table A-6). Clustering was based onsequence similarities and differences in CDR2 and CDR3. Cluster A is themost prominent comprising 50 clones (SEQ ID NO:s 1-50), cluster B andcluster D are each represented by only 1 clone (SEQ ID NO: 51 and SEQ IDNO: 52, respectively), cluster C comprises 4 clones ((SEQ ID NO:s53-56), cluster E comprises 9 clones (SEQ ID NO:s 57-65) and cluster Fcomprises 15 clones (SEQ ID NO:s 66-80).

Sequence variability of the CDRs was determined for the differentclusters. For cluster A, the amino acid sequence of the CDRs of clone117G03 was used as a reference, against which the CDRs of all othercluster A clones were compared. The sequence variability against 117G03is depicted in the tables below.

117G03 Kabat 26 27 28 29 30 31 32 33 34 35 numbering absolute  1*  2  3 4  5  6  7  8  9 10 numbering 117G03 G R T Y R G Y S M G sequence R A FG A Variations *in case position 1 is an R, then position 10 is also A

117G03 Kabat 50 51 52 52a 53 54 55 56 57 58 numbering absolute  1  2  3* 4  5  6  7  8  9 10 numbering 117G03 A I V W S G G N T Y sequence T T DS variations A E A variations P variations *in case position 3 is an T,then position 6 is also E

117G03 Kabat 95 96 97 98 99 100 100a 100b 100c 100d 100e 100f 100g 101102 numbering absolute  1  2  3  4  5  6  7  8  9  10  11  12  13  14 15 numbering 117G03 K I R P Y I F K I A G Q Y D Y sequence T V Pvariations

For cluster B, the amino acid sequence of the CDRs of clone 60E11 isdepicted in the tables below.

60E11 Kabat 26 27 28 29 30 31 32 33 34 35 numbering absolute  1  2  3  4 5  6  7  8  9 10 numbering 60E11 G D I Y K S F D M G sequence

60E11 Kabat 50 51 52 52a 52b 52c 52d 53 54 55 56 57 58 numberingabsolute  1  2  3  4  5  6  7  8  9 10 11 12 13 numbering 60E11 V I G SR G N N R G R T N sequence

60E11 Kabat 95 96 97 98 99 95 101 102 numbering absolute  1  2  3  4  5 6  7  8 numbering 60E11 A P L V A G R P sequence

For cluster C, the amino acid sequence of the CDRs of clone 33G03 wasused as a reference, against which the CDRs of all other cluster Cclones were compared. The sequence variability against 33G03 is depictedin the tables below.

33G03 Kabat 26 27 28 29 30 31 32 33 34 35 numbering absolute  1  2  3  4 5  6  7  8  9 10 numbering 33G03 G R T F S T N P M G sequence

33G03 Kabat 50 51 52 52a 53 54 55 56 57 58 numbering absolute  1  2  3 4  5  6  7  8  9 10 numbering 33G03 A V R W A D G N T F sequence AVariations

33G03 Kabat 95 96 97 98 99 100 100a 100b 100c 100d 100e 100f 100g 101102 numbering absolute  1  2  3  4  5  6  7  8  9  10  11  12  13  14 15 numbering 33G03 G R P W S A Y H S P A E Y V H sequence

For cluster D, the amino acid sequence of the CDRs of clone 11A10 isdepicted in the tables below.

11A10 Kabat 26 27 28 29 30 31 32 33 34 35 numbering absolute  1  2  3  4 5  6  7  8  9 10 numbering 11A10 G R T F S S Y A M A sequence

11A10 Kabat 50 51 52 52a 53 54 55 56 57 58 numbering absolute  1  2  3 4  5  6  7  8  9 10 numbering 11A10 S I S W S G E N T N sequence

11A10 Kabat numbering 95 96 97 98 99 100 100 101 102 a b c d e f g h i jk l absolute 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20numbering 11A10 K I A K T Y P D N W Y W T K S N N Y N Y sequence

For cluster E, the amino acid sequence of the CDRs of clone 52G04 wasused as a reference, against which the CDRs of all other cluster Eclones were compared. The sequence variability against 52604 is depictedin the tables below.

52G04 Kabat 26 27 28 29 30 31 32 33 34 35 numbering absolute 1 2 3 4 5 67 8 9 10 numbering 52G04 G I R I S R N M M G sequence variations N H Tvariations T

52G04 Kabat 50 51 52 53 54 55 56 57 58 numbering absolute 1 2 3 4 5 6 78 9 numbering 52G04 R I T P G G D T Y sequence variations Q S Avariations K

52G04 Kabat 95 96 97 98 101 102 numbering absotute 1 2 3 4 5 6 numbering52G04 Y S T L G S sequence variations R V

For cluster F, the amino acid sequence of the CDRs of clone 50A11 wasused as a reference, against which the CDRs of all other cluster Fclones were compared. The sequence variability against 50A11 is depictedin the tables below.

50A11 Kabat 26 27 28 29 30 31 32 33 34 35 numbering absolute 1 2 3 4 5 67 8 9 10 numbering 50A11 G R S F N M N P L G sequence variations T S T Mvariations A A variations G

50A11 Kabat 50 51 52 52a 53 54 55 56 57 58 numbering absolute 1 2 3 4 56 7 8 9 10 numbering 50A11 A H R W S D G N T Y sequence variations V H SF variations A

50A11 Kabat 95 96 97 98 99 100 100a 100b 100c 100d 100e 100f 100g 101102 numbering absolute 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 numbering50A11 G R P W S A F R S P G E Y V Y sequence variations S Y H A D Ivariations D A T variations N variations S variations K variations R

The clustering based on the sequence transmuted into functionaldifferences (see infra).

4.3 Purification of Monovalent Nanobodies

Since it is not practicable to characterize all clones identified inExample 4.2, representative Nanobodies were selected and expressed in E.coli TG1 as triple Flag, His6-tagged proteins. Expression was induced byaddition of 1 mM IPTG and allowed to continue for 4 hours at 37° C.After spinning the cell cultures, periplasmic extracts were prepared byfreeze-thawing the pellets. These extracts were used as startingmaterial and Nanobodies were purified via IMAC and size exclusionchromatography (SEC).

The Nanobodies were purified to 95% purity as assessed via SDS-PAGE(data not shown).

Example 5 Binding of CD3 Nanobodies to Human TCR/CD3 Expressed on CHO-K1Cells and to Purified Primary Human T Cells

Binding of purified monovalent CD3 Nanobodies to human TCR(2XN9)/CD3expressed on CHO-K1 cells and to purified primary human T cells wasevaluated in flow cytometry as outlined in Example 4.1. Dilution seriesof CD3 Nanobodies 117G03 (cluster A), 60E11 (cluster B), 33G03 (clusterC), 11A10 (cluster D), 52G04 (cluster E) and 50A11 (cluster F) startingfrom 1 μM were applied to the cells.

The results are shown in FIG. 2.

Nanobodies clearly bound to human TCR/CD3 expressed on CHO-K1 cells. Thecluster A representative showed the best affinity, followed by thecluster B, C, F, D and E representatives. Nanobodies bound to purifiedprimary human T cells although with slightly lower potency compared tothe CHO-K1 human TCR(2XN9)/CD3 cells. The representative of cluster Ashowed the best affinity on human primary T cells, in line with the dataon the CHO-K1 (2XN9)/CD3. The EC₅₀ values obtained from the doseresponse curve are represented in Table 1.

TABLE 1 EC50 (M) of anti-CD3 monovalent Nanobodies to CHO-K1 humanTCR(2XN9)/CD3 cells and to purified primary T cells as determined inflow cytometry. CHO-K1TCR(2XN9)/CD3 Primary human T cells Cluster sampleID EC50 (M) 95% LCI 95% UCI EC50 (M) 95% LCI 95% UCI A T0170117G031.8E−09 1.4E−09 2.3E−09 2.2E−08 1.8E−08 2.8E−08 B T0170060E11 1.4E−081.3E−08 1.6E−08 3.4E−08 1.6E−08 7.0E−08 C T0170033G03  >1E−07 / / >1E−07 / / D T0170011A10  >1E−07 / /  >1E−07 / / E T0170052G04 Nobinding  >1E−07 / / F T0170050A11 1.5E−08 3.5E−09 6.3E−08 5.6E−083.4E−08 9.2E−08

Example 6 Determination of Binding Epitope

Binding to human TCR(2IAN)/CD3 expressed on HEK293H cells was evaluatedand compared with the binding to HEK293H human CD3 cells in flowcytometry as outlined in Example 5. Dilution series of CD3 Nanobodiesstarting from 1 μM were applied to the cells. The parental HEK293H cellline was included as TCR/CD3 negative cell line.

The results are shown in FIG. 3.

Binding to HEK293H transfected with human CD3 was observed for allNanobodies. In addition, some Nbs showed binding to HEK293H transfectedwith human TCR/CD3. No binding to the HEK293H parental cell lines wasobserved. The EC₅₀ values obtained from the dose response curve aredepicted in Table 2.

TABLE 2 EC50 (M) of anti-CD3 monovalent Nanobodies to humanTCR(2IAN)/CD3 on human CD3 expressed on HEK293H as determined in flowcytometry. HEK293H CD3 HEK293H TCR(2IAN)/CD3 95% MCF at 95% 95% MCF atCluster sample ID EC50 (M) 95% LCI UCI 1 μM/top EC50 (M) LCI UCI 1μM/top A T0170117G03 NT / / / NT / / / A T0170075G11 8.4E−08 3.8E−081.8E−07 47652 2.0E−08 1.7E−08 2.4E−08 89865 B T0170060E11 6.1E−095.2E−09 7.0E−09 115329 no fit / / 13708 C T0170033G03 2.9E−08 1.1E−087.5E−08 68578 no fit / / 6694 D T0170011A10  >1E−07 / / 3303  >1E−07 / /15882 E T0170052G04  >1E−07 / / 2852  >1E−07 / / 4952 F T0170050A111.5E−09 9.8E−10 2.4E−09 106019 no fit / / 7457

Example 7 Primary Human T Cell Activation Capacity of Purified CD3Reactive Nanobodies

Functionality of purified monovalent CD3 Nanobodies was evaluated in thehuman T cell activation assay. Goat anti-mouse IgG dynabeads (Lifetechnologies #11033) were coated with mouse anti-flag IgG antibodies(Sigma 11804), (15 μg/1E7 beads). After an incubation period of 2 h at4° C., beads were washed and incubated with a fixed (1 μg) concentrationof purified flag tagged Nanobody for 20 min at 4° C. while shaking.Non-coupled Nanobodies were washed away before adding the bead complextogether with soluble mouse anti-CD28 antibody (Pelicluster CD28—Sanquin#M1650) to purified primary human T cells (isolated as described inExample 2.1) from distinct healthy donors.

In addition, the effect of monovalent CD3 binding by the Nanobodies wasevaluated by the incubation of the Nanobody with the purified primaryhuman T cells isolated from distinct healthy donors, without priorcapture onto beads, in the presence of anti-CD28 antibody.

The activation status of the purified primary human T cells wasmonitored by measuring the CD69 expression in flow cytometry usingmonoclonal mouse anti-human CD69PE (BD #557050) after an incubation of24 h at 37° C. and 5% CO2 as described in Example 4.2.

In conclusion, CD3 Nanobodies showed clear CD69 upregulation aftercapturing onto anti-mouse IgG dynabeads (FIG. 4A). None of the CD3reactive Nanobodies, when applied in solution were able to activatepurified primary human T cells as measured by increased expression ofCD69 (FIG. 4B).

Example 8 Binding of Bispecific CD3 Polypeptides to Human T CellReceptor Complex Expressed on Cells

To demonstrate that redirection of engaged T cells to tumour cells ispossible by the Nanobodies, the CD20 antigen was chosen as exemplarytumour target.

Different CD3 building blocks (i.e. Nanobodies) were formatted into abispecific construct with a human CD20 targeting Nanobody (see Table 3).The effector and tumour Nanobodies were genetically linked with 35GSlinker and subsequently expressed in the yeast Pichia according tostandard protocols (bispecific polypeptides).

Irrelevant constructs were generated by replacing the effector or tumourNanobody with an irrelevant anti-egg lysozyme (cablys) Nanobody (Table3).

TABLE 3 Sample ID and description of bispecific constructs Target Nb ×Effector Nb Effector Nb × Target Nb Cluster Clone ID Description CloneID Description C T017000016 20CD019C07-35GS-T0170033G03-FLAG3-HIS6T017000021 T0170033603-35GS-20CD019C07-FLAG3-HIS6 C T01700005920CD019C07-35GS-T0170033G03-FLAG3-HIS6 T017000045T0170033G03-35GS-20CD019C07-FLAG3-HIS6 D T01700001720CD019C07-35GS-T0170011A10-FLAG3-HIS6 T017000022T0170011A10-35GS-20CD019C07-FLAG3-HIS6 / T01700000620CD019C07-5GS-cAbLys3-FLAG3-HIS6 / T01700001820CD019C07-35GS-cAbLys3-FLAG3-HIS6 T017000023cAblys3(D1E)-35GS-20CD019C07-FLAG3-HIS6 C T017000024T0170033G03-35GS-cAbLys3-FLAG3-HIS6 D T017000027T0170011A10-35GS-cAbLys3-FLAG3-HIS6 D T01700006120CD019C07-35GS-T0170011A10-FLAG3-HIS6 T017000047T0170011A10-35GS-20CD019C07-FLAG3-HIS6 F T01700005720CD019C07-35GS-T0170050A11-FLAG3-HIS6 T017000043T0170050A11-35GS-20CD019C07-FLA63-HIS6 E T01700005620CD019C07-35GS-T0170052G04-FLAG3-HIS6 T017000048T0170052G04-35GS-20CD019C07-FLAG3-HIS6 A T01700006620CD019C07-35GS-T0170061D09-FLAG3-HIS6 T017000072T0170061D09-35GS-20CD019C07-FLAG3-HIS6 A T01700007120CD019C07-35GS-T0170061F07-FLAG3-HIS6 T017000034T0170061F07-35GS-20CD019C07-FLAG3-HIS6 A T01700005220CD019C07-35GS-T0170061F04-FLAG3-HIS6 T017000039T0170061F04-35GS-20CD019C07-FLAG3-HIS6 A T01700006220CD019C07-35GS-T0170117G03-FLAG3-HIS6 T017000036T0170117G03-35GS-20CD019C07-FLAG3-HIS6 B T01700005320CD019C07-35GS-T0170060E11-FLAG3-HIS6 T017000040T0170060E11-35GS-20CD019C07-FLAG3-HIS6

Binding of the bispecific constructs to human TCR/CD3 expressed onCHO-K1 cells, purified primary human T cells and human CD20 positiveRamos cells (ATCC: CRL-1596) was evaluated in flow cytometry as outlinedin Example 5 and is presented in FIG. 5.

The EC₅₀ values obtained from the dose response curve are depicted inTable 4.

TABLE 4 EC50 (M) of anti-CD3 monovalent Nanobodies to CHO-K1 humanTCR(2XN9)/CD3, primary human T cells and Ramos cells as determined inflow cytometry. CHO-K1 huTCR(2XN9)/CD3 hu T cells Ramos EC50 95% EC5095% EC50 95% Cluster sample ID (M) 95% LCI UCI (M) 95% LCI UCI (M) 95%LCI UCI A T017000062 1.5E−07 1.1E−07 2-1E−07 8.9E−08 7.2E−08 1.1E−071.9E−09 1.8E−09 2.1E−09 A T017000036 7.6E−09 5.9E−09 9.6E−09 3.5E−082.8E−08 4.4E−08 2.1E−08 1.9E−08 2.3E−08 B T017000053 1.5E−07 1.3E−071.7E−07  >1E−07 / / 1.8E−09 1.6E−09 2.0E−09 B T017000040 1.6E−07 1.5E−071.8E−07  >1E−07 / / 3.2E−08 2.9E−08 3.7E−08 C T017000016  >1E−07 / / >1E−07 / / 1.5E−09 1.4E−09 1.6E−09 C T017000021  >1E−07 / / 5.4E−084.0E−08 7.3E−08 4.7E−09 4.4E−09 5.0E−09

The data indicate similar binding of the CD3×CD20 bispecific Nanobody(dotted line) compared to their monovalent counterparts, but a reducedbinding of the CD20×CD3 bispecific Nanobody (full line) compared totheir monovalent counterparts to CMO-K1 human TCR(2XN9)/CD3 cells and toprimary human T cells. On the human CD20 Ramos cell, the bispecific CD3Nanobodies with the CD20 at the C terminus showed reduced binding.

Example 9 Functional Characterization of Bispecific CD20×CD3Polypeptides in Flow Cytometry Based Killing Assay

In order to assess whether bispecific polypeptides were able to killtumour cells, cytotoxicity assays were performed with isolated human Tcells as effector cells.

Human T cells were isolated as described in Example 2.1. The quality andpurity of the purified human T cells was checked with anti-CD3(eBioscience #12-0037-73); anti-CD8 (BD Bioscience #345775); anti-CD4(BD Bioscience #345771); anti-CD45RO (BD Bioscience #555493);anti-CD45RA (BD Bioscience #550855) and anti-CD19 (BD Bioscience#555413), anti-CD25 (BD Pharmigen #557138), anti-CD69 (BD Pharmigen#557050) fluorescently labelled antibodies in a flow cytometric assay.Human CD20 expressing Ramos cells and human CD20 expressing Raji cells(ECACC: 85011429), labelled with the PKH-26 membrane dye as describedabove were used as target cells. 2.5×10⁵ effector and 2.5×10⁴ targetcells were co-incubated in 96-well V-bottom plates at an effector versustarget ratio of 10:1. For measurement of the concentration-dependentcell lysis, serial dilutions of bispecific polypeptides (Table 3) wereadded to the samples and incubated for 18 h in a 5% CO₂ atmosphere at37° C. After incubation, cells were pelleted by centrifugation andwashed with FACS buffer. Subsequently, cells were resuspended in FACSbuffer supplemented with 5 nM TOPRO3 (Molecular Probes cat# T3605) todistinguish live from dead cells. Cells were analysed using a FACS Arrayflow cytometer (BD Biosciences). Per sample, a total sample volume of 80μl was acquired. Gating was set on PKH26 positive cells, and within thispopulation the TOPRO3 positive cells were determined.

The CD3 bispecific polypeptides showed dose dependent killing of theRamos cells (FIG. 6A). T017000045 (cluster C,T0170033G03-35GS-20CD019C07-FLAG3-HIS6) showed a dose dependent killingon both Ramos (FIG. 6A) and Raji (FIG. 6B) cells confirming that theobserved cytotoxic effect was not restricted to a single tumour cellline. The expression level of the tumour antigen, CD20, was determinedfor both cell lines (FIG. 7).

The IC₅₀ values and the % lysis obtained from the dose response curveare depicted in Table 5 (% lysis=% death cells at 500 nM of Nanobodyminus % dead cells of the no Nanobody control).

TABLE 5 Average IC50 (M) of the bispecific constructs in the flowcytometry based purified primary human T cell mediated Ramos killingassay using an effector to target ration of 10 to 1. ID monovalent IDconstruct % % lysis ID construct % % lysis Cluster Nb (CD20xCD3) n IC50(M) lysis (stdev) (CD3xCD20) n IC50 (M) lysis (stdev) A T0170117G03T017000062 6 3.9E−09 22 7 T017000036 3 5.5E−09 26 6 B T0170060E36T017000053 2 7.0E−09 22 3 T017000040 3 6.8E−09 29 5 C T0170033G03T017000016 2 9.2E−09 4 2 T017000045 1 / 0 D T0170011A10 T017000061 22.9E−08 13 0 T017000047 0 E T0170052G04 T017000056 2 1.5E−08 4 6T017000048 0 F T0170050A11 T017000057 2 1.4E−09 3 4 T017000043 0

These results demonstrate that the CD3 bispecific polypeptides caninduce T cell mediated killing of tumour target positive cell lines.When either the targeting Nanobody or the effector Nanobody was replacedby an irrelevant no effect on the viability of the Ramos cells could beobserved. There was no clear preference of the orientation between theindividual binding blocks in the bispecific polypeptide.

Example 10 Functional Characterization of Bispecific CD20×CD3Polypeptides in an xCELLigence Based Killing Assay

The CD3 multispecific polypeptides (Table 3) were also tested for theircell toxicity on human CD20 transfected adherent target cells in thepresence of human effector T cells using real-time electrical impedancebased technique. Here, fluctuations in impedance induced by theadherence of cells to the surface of an electrode were measured. T cellsare non-adherent and therefore do not impact the impedance measurements.

In brief, the xCELLigence station was placed in a 37° C. incubator at 5%CO₂. 50 μl of assay medium was added to each well of E-pate 96 (ACEABiosciences; cat#05 232 368 001) and a blank reading on the xCELLigencesystem was performed to measure background impedance in absence ofcells. Subsequently, human CD20 transfected CHO-K1 or CHO-K1 parentalcells (1×10⁴) were seeded onto the E-plates 96, and 50 μl of a serialdilution of Nanobody was added. After 30 min at RT 50 μl of human Tcells were added per well (3×10⁵) to have an effector to target ratio of30:1. The plate was placed in the xCELLigence station and impedance wasmeasured every 15 min during 3 days. The data were analysed 44 h afterstart of the assay.

The IC₅₀ values are depicted in Table 6.

The multispecific polypeptides showed tumour antigen dependent killing,they induced a dose dependent human T cell mediated killing of the humanCD20 transfected CHO-K1 cells (FIG. 8) but the constructs were not ableto induce T cell mediated killing of CHO-K1 parental cells (FIG. 9).

TABLE 6 Average IC50 (M) of the multispecific constructs in thexCELLigence based human T cell mediated CHO- K1 CD20 killing assay usingan effector to target ration of 30 to 1, analysed at 44 h after seeding.sample ID sample ID Cluster ID monovalent Nb (CD20xCD3) n IC50 (M)(CD3xCD20) n IC50 (M) A T0170117G03 T017000062 5 7.88E−10 T017000036 21.04E−09 B T0170060E11 T017000053 2 2.81E−10 T017000040 2 4.89E−10 CT0170033G03 T017000016 3 6.81E−12 T017000021 2 1.52E−10

These results confirm the outcome obtained in the flow cytometry basedkilling assay of Example 9. In addition, only when the tumour targetantigen is present T cell mediated killing was observed, indicating thatthe bispecific polypeptides are critically dependent on their target forinduction of cytotoxicity.

Example 13 Linker Length Evaluation of the Bispecific Polypeptides

To evaluate the impact of the linker length used in the CD20/CD3bispecific polypeptides on the cytotoxic capacity, the effector andtumour Nanobodies were genetically linked with a 5GS, 9GS or 35GS linkerand subsequently expressed in Pichia according to standard protocols(see Table 7).

TABLE 7 Sample ID and description of bispecific constructs to evaluateimpact of linker length Target Nb × Effector Nb Effector Nb × Target NbCluster Clone ID Description Clone ID Description C T01700000420CD019C07-5GS-T0170033G03-FLAG3-HIS6 D T01700000520CD019C07-5GS-T0170011A10-FLAG3-HIS6 C T01700001020CD019C07-9GS-T0170033G03-FLAG3-HIS6 T017000020T0170033G03-9GS-20CD019C07-FLAG3-HIS6 D T01700001120CD019C07-9GS-T0170011A10-FLAG3-HIS6 T017000026T0170011A10-9GS-20CD019C07-FLAG3-HIS6 C T01700001620CD019C07-35GS-T0170033G03-FLAG3-HIS6 T017000021T0170033G03-35GS-20CD019C07-FLAG3-HIS6 D T01700001720CD019C07-35GS-T0170011A10-FLAG3-HIS6 T017000022T0170011A10-35GS-20CD019C07-FLAG3-HIS6 / T01700000620CD019C07-5GS-cAbLys3-FLAG3-HIS6 / T01700001220CD019C07-9GS-cAbLys3-FLAG3-HIS6 / T01700001820CD019C07-35GS-cAbLys3-FLAG3-HIS6 T017000023cAbLys3(D1E)-35GS-20CD019C07-FLAG3-HIS6 C T017000024T0170033G03-35GS-cAbLys3-FLAG3-HIS6 D T017000027T0170011A10-35GS-cAbLys3-FLAG3-HIS6

The impact of the linker length used in the CD20/CD3 bispecificpolypeptides on the human primary effector T cell induced cellulartoxicity on the adherent CHO-K1 human CD20 target was evaluated usingreal-time electrical impedance based technique as described in Example9.

The results are summarized in FIG. 10.

All bispecific polypeptides, i.e. ail linker lengths demonstratedspecific cell killing. Little difference in potency was observed betweenthe different linkers 9GS and 35GS linker for these bispecificpolypeptides.

Example 12 Effector to Target Ratio Evaluation of the CD3 BispecificPolypeptides

To evaluate the effect of different effector to target (E:T) ratios onthe killing properties of the Nanobodies, CD20×CD3 bispecificpolypeptides were incubated with 2.5×10⁴ PKH labelled Ramos cells in thepresence of respectively 2.5×10⁵ (E:T=10:1), 1.25×10⁵ (E:T=5:1), 5×10⁴(E:T=2:1), 2.5×10⁴ (E:T=1:1), human T cells as described Example 2.

Exemplary results are shown in FIG. 11. The IC₅₀ values are depicted inTable 8.

TABLE 8 IC50 (M) of the bispecific constructs in the flow cytometrybased T cell mediated Ramos killing assay using different effector totarget ratio's. sample ID Cluster ID monovalent Nb (CD20xCD3) E:T n IC50(M) 95% LCI 95% UCI % lysis A T0170117G03 T017000062 10 1 7.3E−095.2E−09 1.0E−08 15 A T0170117G03 T017000062 5 1 8.7E−09 5.3E−09 1.4E−0810 A T0170117G03 T017000062 2 1 7.1E−09 3.1E−09 1.6E−08 6 A T0170117G03T017000062 1 1 2.5E−08 1.9E−09 3.3E−07 2

The bispecific construct induced killing of the human CD20 target cellsat different E:T ratios, even at a ratio of 1:1, after an incubationtime of 18 h with little difference in potency. Although there is animpact on the E:T ratio on the % lysis, this can also be linked to theincubation time (see below).

Example 13 Time Dependent Cytolytic Activity of CD20/CD3 BispecificConstructs in the Purified Primary Human T Cell Mediated Assay inxCELLigence

To evaluate the impact of incubation time on the killing properties ofthe CD20×CD3 bispecific constructs, specific lysis of target cells wascalculated for different time-points in xCELLigence. In brief, thexCELLigence station was placed in a 37° C. incubator at 5% CO₂. 50 μl ofassay medium was added to each well of E-pate 96 (ACEA Biosciences;cat#05 232 368 001) and a blank reading on the xCELLigence system wasperformed to measure background impedance in absence of cells.Subsequently, human CD20 transfected CHO-K1 or CHO-K1 parental cells(1×10⁴) were seeded onto the E-plates 96. After 20 h, purified primaryhuman T cells (described supra) and either 100 nM or 1.5 nM bispecificconstructs were added, respectively. The cell index (CI) was measuredevery 15 min during 5 days. Using the normalized CI (The normalized CellIndex—NCI, is calculated by dividing the Cell Index value at aparticular time point by the Cell Index value of the time-point whenpurified primary human T cells were added) specific lysis at differenttime points of the condition with constructs was calculated in relationto the condition lacking construct. (% specificlysis=((NCI_(no construct)−NCI_(with construct))/NCI_(no construct)))×100.

The results are depicted in FIG. 12.

Already one hour after the addition of human primary T cells and themultispecific construct, an increase of the cell index can be observedwhich clearly increased further upon longer incubation times. Themaximal effect was clearly dependent on the incubation time but theobtained IC50 value did not change with increased incubation times. Theirrelevant construct did not show any killing of the human CD20 CHO-K1cells.

Example 14 Exploration of Half-Life Extension

It was hypothesized that FILE via albumin binding might be suitable tocomply with various requirements, including (i) half-life extension(HLE) of the moiety; and (ii) efficacy of the multispecific. Preferably,the HLE function would not impair the penetration of tumours andtissues.

Alb11, a Nanobody binding to human serum albumin (HSA) was linked to themultispecific CD20×CD3 polypeptides to increase the in vivo half-life ofthe formatted molecules (WO 06/122787). A number of formats weregenerated based on the CD20 tumour targeting building block at theN-terminus, the CD3 recruiting building blocks in the middle and thealbumin targeting Nanobody at the C-terminus using a 35GS linker andexpressed as indicated above. An overview of the explored formats isshown in Table 9.

TABLE 9 Sample ID and description of HLE constructs Cluster Sample IDDescription A T017000094 20CD019C07-35GS-T0170117G03-35GS-ALB11-FLAG3-HIS6 B T017000096 20CD019C07-35GS-T0170060E11-35GS-ALB11-FLAG3-HIS6

As the binding of HSA to the Alb11 Nanobody might have an impact on theaffinity or potency of the HLE constructs, the half-life extendedNanobodies were characterized for binding to CD3 overexpressing CHO-K1and primary human T cells. In addition, the potency in the functional Tcell dependent Ramos B cell killing assay was evaluated in the presenceand absence of HSA (described in 14.1 and 14.2 below).

14.1 Impact of Human Serum Albumin on the Binding Properties

Analogous to the experiments described in Example 5, binding ofhalf-life extended anti-CD3 multispecific constructs to CHO-K1 humanTCR(2XN9)/CD3 cells, primary human T cells and Ramos cells was evaluatedin a flow cytometric assay.

The results are provided in FIG. 13. The EC₅₀ values obtained in thisassay are listed in Table 10.

TABLE 10 Table with EC50 values of cell based binding to CHO-K1 humanTCR(2XN9)/CD3, primary human T cells and Ramos cells as determined inflow cytometry CHO-K1-K1 huTCR (2XN9)/CD3 hu T cells Ramos EC50 EC50EC50 Cluster sample ID (M) 95% LCI 95% UCI (M) 95% LCI 95% UCI (M) 95%LCI 95% UCI A T017000094  >1E−07 / /  >1E−07 / / 1.9E−09 1.7E−09 2.1E−09B T017000096 1.6E−07 1.4E−07 1.8E−07 3.2E−08 2.0E−08 5.3E−08 1.7E−091.5E−09 1.9E−09

Comparison of the CD20-35GS-CD3 HLE construct with the non-HLEconstructs showed similar binding on all three cell lines tested. Thedata presented showed that coupling of the Alb11 building block did notinfluence the binding properties.

14.2 Impact of Human Serum Albumin on Potency in Human T Cell Mediated BCell Killing Assay

The functionality of half-life extended anti-CD3 Nanobodies (Table 9)was evaluated in the human T cell mediated Ramos killing assay asdescribed in Example 10 in the presence and absence of 30 μM HSA andcompared with the functionality of the non-HLE bispecific constructs.

The results are depicted in FIG. 14. The IC₅₀ values obtained in thisassay are listed in Table 11.

TABLE 11 Table with IC50 and % lysis of the CD20 × CD3 Nanobodies in theT cell dependent B-cell (Ramos) killing assay to evaluate the effect ofHLE EC50 95% 95% % Cluster sample ID (M) LCI UCI lysis A T0170000626.4E−10 3.5E−10 1.2E−09 15 A T017000094 3.2E−09 1.7E−09 6.1E−09 14 AT017000094 2.0E−09 1.2E−09 3.3E−09 14 A T017000094 + HSA 2.0E−08 7.0E−095.5E−08 7 B T017000053 1.8E−09 8.9E−10 3.8E−09 12 B T017000096 3.8E−092.1E−09 7.0E−09 15 B T017000096 2.6E−09 1.5E−09 4.7E−09 16 BT017000096 + HSA 4.8E−09 1.1E−09 2.0E−08 6

The results indicate that the inclusion of the albumin targeting Nb inthe construct as such did not have an essential impact on the obtainedpotency or efficacy. Although a minor loss of efficacy/potency wasobserved in the presence of HSA, the half-life extended CD3 bispecificpolypeptides were still potent in tumour cell killing.

Example 15 Functional Characterization of Multispecific Polypeptides inan xCELLigence Based Human T Cell Mediated HER2-Positive Tumour KillingAssay

In order to assess the general applicability of the CD3 building blocksin directing T cells to tumour cells, CD3 building blocks were combinedwith a different TAA, in this case a Nanobody binding to HER2.

The anti-CD3 building block was combined with a Nanobody against theHER2 solid tumour antigen in two orientations (Table 12) andcharacterized in the xCELLigence based human T cell mediatedHER2-Positive Tumour Killing assay as described in Example 10 using twoHER2 expressing cell lines: 5K-BR-3 (ATCC: HTB-30), MCF-7 (ATCC: HTB-22)and a HER2 negative reference cell line, MDA-MB-468 (ATCC HTB-132) astarget cell population. Human HER2 expression levels were confirmedusing the monovalent anti-HER2 Nanobody HER2005F07 in flow cytometry asdescribed in Example 9 using 100 nM of anti-HER2 Nanobody and shown inFIG. 15.

TABLE 12 Sample ID and description of HER2/CD3 constructs Cluster SampleID Description A T017000101 HER2005F07(Q108L)-35GS-T0170117G03-FLAG3-HIS6 A T017000100 T0170117G03-35GS-HER2005F07(Q108L)- FLAG3-HIS6

In brief, SKBR3 (4×10⁴ cells/well), MDA-MB-468 (4×10⁴ cells/well) orMCF-7 (2×10⁴ cells/well) were seeded in 96 well E-plates and incubatedwith 6×10⁵ cells and 3×10⁵ cells human primary T cells (effector targetratio of 15 to 1) in the presence or absence of multispecific constructsand followed over time. Data were analysed after 18 h after start of theexperiment and shown in FIG. 16.

The IC₅₀ values obtained in this assay are listed in Table 13.

TABLE 13 Overview IC50 of the HER2/CD3 constructs in the T cell mediatedHER2-Positive Tumour Killing assay xCELLigence based cytotoxicity(readout 18 h)-SKBR3 (E/T = 15:1) ID monovalent ID construct IC50 95%95% ID Construct IC50 95% 95% Cluster Nb (HER2xCD3) n (M) LCI UCI(CD3xHER20) n (M) LCI UCI A T0170117G03 T017000101 1 2.6E−11 2.2E−113.1E−11 T017000100 1 9.5E−12 8.12E−12 1.1E−11 xCELLigence basedcytotoxicity (readout 18 h)-MCF-7 (E/T = 15:1) ID monovalent IDconstruct IC50 95% 95% ID Construct IC50 95% 95% Cluster Nb (HER2xCD3) n(M) LCI UCI (CD3xHER20) n (M) LCI UCI A T0170117G03 T017000101 1 1.1E−107.3E−11 1.8E−10 T017000100 1 6.9E−11 4.5E−11 1.0E−10 xCELLigence basedcytotoxicity (readout 18 h)-MDA-MB-468(E/T = 15:1) ID monovalent IDconstruct IC50 95% 95% ID Construct IC50 95% 95% Cluster Nb (HER2xCD3) n(M) LCI UCI (CD3xHER2) n (M) LCI UCI A T0170117G03 T017000101 1 / / /T017000100 1 / / /

The data indicate specific killing of HER2 positive tumour cell lines bydirecting human primary T cells to the tumour cells via the CD3Nanobody. Hence, the CD3 building blocks are broadly applicable fordirecting CTLs to tumours. Despite the large difference in tumourantigen density on SKBR3 and MCI-7 cells, both are efficiently killed bythe addition of multispecific Nanobody constructs.

Example 16 Effect of CD20/CD3 Polypeptides on IFN-γ Release by Human TCells in the xCELLigence Killing Assay

The CD20×CD3 polypeptides were evaluated for their capacity to inducecytokine secretion in the human T cell mediated CHO_K1 human CD20killing assay based on xCELLigence as described in Example 10. Therelease of the cytokine IFN-γ was measured by ELISA. Briefly, CHO-K1human CD20 cells were seeded in 96 E plate and after 20 h, purifiedhuman primary T cells with or without bispecific CD20×CD3/irrelevantconstructs were added to the E plates as described in Example 13. 72 hafter the addition of the human primary T cells/constructs to theE-plates, IFN-γ production by the human primary T cells was measured.Maxisorp 96-well ELISA plates (Nunc) were coated with anti-human IFN-γantibody (BD Bioscence #551221). After overnight incubation, plates werewashed and blocked with PBS+2% BSA for 1 h at room temperature. Next,plates were incubated with 100 μl of the supernatants (2 fold diluted)and 1 μg/ml biotinylated anti-human IFN-γ antibody (BD Bioscience,#554550) 2 h 30 min while shaking, washed again and incubated withstreptavidin-HRP (Dakocytomation #P0397). After 30 min, TMB One Solution(Promega #G7431) was added. The reaction was stopped with 2M H2SO4 andthe Nanobody dose dependent production of IFN-γ was determined bymeasurement the OD at 405 nm using the Tecan sunrise 4.

The results are shown in FIG. 17. The EC₅₀ values obtained in this assayare listed in Table 14.

TABLE 14 EC50 (M) of the CD20/CD3 dependent IFN-γ secretion by human Tcells in the human T cell mediated xCELLigence based killing assay. IDID construct monovalent Nb (CD20 × CD3) n EC50 (M) 95% LCI 95% UCIT0170117G03 T017000062 1 6.2E−10 5.3E−10 7.5E−10 T0170060E11 T0170000531 9.1E−10 7.2E−10 1.1E−09

The bispecific CD20/CD3 polypeptide showed a dose dependent productionof IFN-γ. Incubation with the irrelevant construct or the conditionwithout bispecific construct did not induce any IFN-γ production.

Example 17 In Vivo Proof-of-Concept in a PBMC B Cell Depletion Model

In this B-cell depletion model, human PBMC were injectedintraperitoneally to NOG mice. PBMC-derived B cell killing bypolypeptide-mediated recruitment of T cells present in the PBMCpopulation was evaluated reflecting the potential of multispecificpolypeptides to activate T cells by direct linkage of T cells via CD3 totarget B cells via CD20, resulting in target cell killing.

The in vivo efficacy of a CD3/CD20 multispecific polypeptides(T017000084, CD20×CD3 binding, cluster A) on B cell depletion in a PBMCNOG mouse model was evaluated and compared with the irrelevantpolypeptide 1017000088. The study demonstrated a clear effect onPBMC-derived B cell depletion in spleen.

In detail, the B cell depletion was evaluated in mice, intraperitoneallyinjected with 3×10⁷ PBMCs in 500 μL of PBS at day 3 (D3) after a wholebody irradiation of mice with a γ-source (1.44 Gγ, 60Co) on day 0 (D0)and randomization of the mice into groups each of 12 animals. Thetreatment started on D3 one hour after PBMC injection and was repeatedfor 5 consecutive days, in total until day 7 (D7) (FIG. 18). One doselevel of the CD3/CD20 binding NB was tested (24 mg/kg).

On day 18 (D18), mice were sacrificed and the spleen was collected forFACS analysis (mCD45, hCD45, hCD19, hCD20) to analyze and quantify thepresence of PBMC-derived human B cells (hCD19+hCD20+hCD45+mCD45−).

Results for PBMC-derived B cell depletion are represented in FIG. 19. Inthe spleen, B cell counts in the group treated with 1017000084 wereclearly different from the irrelevant polypeptide-treated group at thedose level tested. The dose is estimated to be on the maximum effect.

In conclusion, these results demonstrate that CD3/CD20 bindingmultispecific polypeptides are clearly able to decrease PBMC-derived Bcells in spleen in this model. This confirmed the polypeptide-induced Tcell activation by cross-linking T cells to target B cells and killingof the latter.

Example 18: In Vivo Proof-of-Concept in a Ramos B-Cell Depletion Model

In this B-cell depletion model, Ramos cells (a Burkitt's lymphoma cellline) and human PBMC were injected respectively intravenously andintraperitoneally to NOG mice. Ramos B cell and PBMC-derived B cellkilling by polypeptide-mediated recruitment of T cells present in thePBMC population was evaluated reflecting the potential of multispecificpolypeptides to activate T cells by direct linkage of T cells via CD3 totarget B cells via CD20, resulting in target cell killing.

The in vivo efficacy of the multispecific polypeptide T017000084(CD20×CD3 binding) on B cell depletion in a Ramos NOG mouse model wasevaluated and compared with the irrelevant bispecific polypeptide1017000088 (irrelevant Nanobody+CD3 binding Nanobody). The studydemonstrated a statistically significant effect in bone marrow andspleen on Ramos B-cell depletion and on PBMC derived B cell depletion inspleen.

In detail, the B cell depletion was evaluated in mice, intravenouslyinjected with 10⁶ Ramos cells in 200 μL of Roswell Park MemorialInstitute (RPM′) medium 1640 at day one (01). This injection took place24 hours after a whole body irradiation of mice with a γ-source (1.44Gγ, 60Co) (D0). 10⁷ PBMCs (500 μL in PBS) were injected on D3 (i.e. twodays after tumor cell injection) after randomization of the mice intogroups each of 12 animals. The treatment started on D3 one hour afterPBMC injection and was repeated for 5 consecutive days in total until D7(FIG. 20). One dose level of the CD3/CD20 binding polypeptide was tested(24 mg/kg).

On D20 or on D21, mice were sacrificed and spleen and bone marrow(femur) were collected for FACS analysis (mCD45, hCD45, hCD19, hCD20,hCD10) to analyze and quantify the presence of Ramos B cells(hCD19+hCD20+hCD45+mCD45-hCD10+) and PBMC-derived B cells(hCD19+hCD20+hCD45+mCD45−hCD10−).

Results for Ramos B cell depletion are represented in FIG. 21. Micetreated with an irrelevant bispecific polypeptide were considered ascontrol group for analyses. Statistical analysis has been performed withF-tests from the mixed-effects ANOVA analysis. For the bone marrow andspleen, B cell counts in the group treated with T017000084 werestatistically significantly different from the irrelevantpolypeptide-treated group at the tested dose level. In the spleen, thedose is close to or estimated to be on the maximum effect.

Results for PBMC-derived B cell depletion are represented in FIG. 22.Both in bone marrow and in spleen, a statistically significantdifference in human B cell numbers was seen for T017000084 versus theirrelevant polypeptide at the tested dose level. The dose is estimatedto be on the maximum effect.

In conclusion, these results demonstrate that CD3/CD20 bispecificpolypeptides are able to significantly decrease Ramos B cells andPBMC-derived B cells in spleen and bone marrow in this model. Thisconfirms the Nanobody-induced T cell activation by cross-linking T cellsto target B cells and killing of the latter.

Example 19 Targeting of Tumour Cells with Multispecific T Cell EngagingPolypeptides

The therapeutic activity of T cell engaging strategy can be improved bythe simultaneous targeting of multiple tumour associated antigens. Oftentumour cells create an escape mechanism by the down-regulation oftargeted antigens within a therapy. The simultaneous targeting ofmultiple antigens is likely to reduce the probability of generatingtumour escape variants. The individual affinity of the respective tumourtargeting Nanobodies may be varied such that preferable binding toeither a single marker or simultaneous binding to both tumour markers isachieved. Antigens present on different cell populations can be combinedor even soluble proteins can be targeted in combination with a tumourassociated antigen.

As the Nanobody platform is ideally suited to combine differentspecificities into a multispecific format, the CD3 Nanobodies of theinvention are combined into formats illustrating these concepts, i.e.with different tumour antigen binding Nanobodies in a multispecificpolypeptide.

For the double tumour antigen targeting concept, a Nanobody reactivetowards a first tumour antigen (TA1, e.g. CEA) is linked to a secondNanobody with different specificity (TA2, e.g. EGFR), different fromTA1, in combination with a CD3 reactive Nanobody. The specific order ofthe building blocks is varied within the format as well as the appliedlinker lengths in between the different building blocks. Combinations ofTA1 and TA2 which are tested are depicted in Table 15.

TABLE 15 Combination of CD3, TA1, TA2 and Alb binding building blocks inmultispecific polypeptides. T-cell ISV TA1 ISV TA2 ISV ALB-ISV CD3 CEAIrr + CD3 CEA Irr − CD3 CEA EGFR + CD3 CEA EGFR − CD3 Irr EGFR + CD3 IrrEGFR −

In order to test half-life extension, an albumin binding Nanobody isincluded as well in the polypeptides as set out in Table 15.

To demonstrate the specific killing, a mixed cell culture assay systemis used where TA1 single positive (e.g. MC38-huCEA or MKN45) and TA2single positive tumour cells (e.g. Hela or Her14) are co-incubated. Theexpression level of the respective tumour antigens was determined indifferent cell lines and is represented in FIG. 23. Upon addition of thepolypeptides of the invention, primary human T cells and albumin ifrequired, the T cell mediated cytotoxicity is monitored based on acytometric read out. A comparison is made with respect to doublenegative cells or formats containing one or more irrelevant Nanobodies.

In order to verify the specific killing, the induced killing of doublepositive tumour (for TA1 and TA2, e.g. L5174T or LoVo) cells is comparedwith the induced killing of single positive tumour cells. For this, a Tcell mediated cytotoxicity assay is used as described above with asingle type of tumour cells positive for both markers (cf. Example 17).

Example 20 Targeting of Tumour Cells with Multispecific T Cell EngagingPolypeptides

As mentioned above, the therapeutic activity of T cell engaging strategycan be improved by the simultaneous targeting of multiple tumourassociated antigens. Not only tumour cells create an escape mechanism bythe down-regulation of targeted antigens within a therapy, but also byintroducing (point-)mutations. Also in this case, simultaneous targetingof multiple epitopes on an antigen is likely to reduce the probabilityof generating tumour escape variants. Moreover, targeting multipleepitopes on a single antigen can increase the affinity of binding(avidity effect).

As the Nanobody platform is ideally suited to combine differentspecificities into a multivalent format, the anti-CD3 Nanobodies of theinvention are combined into formats illustrating these concepts, i.e.with different tumour antigen binding Nanobodies in a multispecificpolypeptide.

For the multivalent tumour antigen targeting concept, two Nanobodiesreactive towards an antigen are linked (TA1 and TA2, respectively),followed by a CD3 reactive Nanobody. The specific order of the buildingblocks is varied within the format as well as the applied linker lengthsin between the different building blocks. Combinations of TA1 and TA2which are tested are depicted in Table 16.

TABLE 16 Combination of CD3, TA1, TA2 and Alb binding building blocks inmultispecific polypeptides. T-cell ISV TA1 ISV TA2 ISV ALB-ISV CD3EGFR-1 (7D12) EGFR-2 (9G08) + CD3 EGFR-1 (7D12) EGFR-2 (9G08) − CD3Her2-1 (5F07) Her2-2 (47D05) + CD3 Her2-1 (5F07) Her2-2 (47D05) −

In order to test half-life extension, an Alb Nanobody is included aswell in the polypeptides as set out in Table 16.

The potency and efficacy of these multivalent formats is evaluated andcompared with the respective bispecific formats in an in vitro tumourcell killing assay comparable to the assay described in Example 10 butwith the relevant cell lines (e.g. Hela, Her14, Ls174T, SKBR3, MCF7).Additionally, the effector-target ratio is varied such that an estimateis made whether a multivalent/multispecific polypeptide has a higherefficacy with lower effector target ratios.

TABLES

TABLE A-1 Sequence alignment of CD3 cluster A binders T0170PMP117G03:EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVEGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTVTVSST0170PMP061F04:..................................A....S..............D.......F..........S....L.....N.....T.L..............................T0170PMP081D02:..................................A....S..............D.......F..........SV...L.....N.....T.L..............................T0170PMP120D07:..................................A...................D.......F..........S....L.....N.E...T.L..............................T0170PMP118D11:..................................A...................D.......F..........S....L.....N.E.G.T.L........................L.....T0170PMP062A11:..................................A...................D.......F..........S....LS.H..N.....T.V..............................T0170PMP118A08:...P..............................A...................D.......F..........S....LS.H..N.....T.V..............................T0170PMP122C07:..................................A...................D.......F..........S....LS.H.AN.....T.V........................L.....T0170PMP062D09:.............S.........N..............................D..S....F..........S...I......N.....T.V........................L.....T0170PMPC62G08:.......................N..........A....S.....G.....T..E..A....F...............L.....N.....T.L........................L.....T0170PMP126E04:.......................N..........A....S...........T..E..A....F...............L.....N.....T.L........................L.....T0170PMP116E01:.......................N..........A....S...........T..E..A....F.R.............L.....N.....T.L........................L.....T0170PMP117E03:.......................N.R........A....S...........T..E..A....F...............L.....N.....T.L........................L.....T0170PMP062B10:.......................S..........A....S...........T..E..A....F...............L.....N.....T.L........................L.....T0170PMP118E11:.......................N..........A....S...........T..E..A....F...............L.....N.....T.L......T.......................T0170PMP075A10:.......................D..........A....S...........T..E..A....F...............L.....N.....T.L........................L.....T0170PMP062C06:.......................N........G.A....S...........T..E..A....F...............L.....N.R...T.L........................L.....T0170PMP112G06:.......................N..........A....S...........T..E..A....F...............L..R..N.E...T.L........................L.....T0170PMP061A09:..........L.......T....N..........A....P...........T..E..P....F...............L..R..N.E...T.L........................L.....T0170PMP061B06:..................T....N..........A....P...........T..E..P....F...............L..R..N.E...T.L........................L.....T0170PMP111C01:..................T....N..........A....P...........T..E..P....FA..............L..R..R.E...T.L........................L.....T0170PMP061D06:..................T....N...A......A....P...........T..E..P....F...............L..R..R.E...T.L..............................T0170PMP061E09:..................T....N..........A....P...........T..E..P....F...............L..R..R.E...T.L.............V................T0170PMP078A07:..................T....N.R........A....P...........T..E..P....F...............L..R..R.E...T.L..............................T0170PMP061A04:.............T........V...........A....P...........T..E.......F...............L.....G.....T.L..............P.........L.....T0170PMP115A03:.............T........V...........A....P...........T..E.......F...............L...........T.L..............P.........L.....T0170PMP061F09:...P.........T........V...........A....P...........T..E.......F...............L...........T.L..............P.........L.....T0170PMP112D04:.............T........V...........A....P....G......T..E.......F...............L...........T.L..............P.........L.....T0170PMP114E06:.............T........V.P.........A....P...........T..E.......F...............L...........T.L..............P.........L.....T0170PMP113G04:.............T........V...........A....P...........T..E.......F...............L....A......T.L..............P.........L.....T0170PMP061D09:..........L..T........V...........A....P...........T..E.......F...............L...........T.L..............P...............T0170PMP062G05:.............T........V...........A....P...........T..E.......F.............S.L...........T.L..............P...............T0170PMP114C05:.............T........V...........A....P...........T..E.......L...............L...........T.L..............P...............T0170PMP117G05:.............T........V...........A....P...........T..E.......F...............L...........T.L.............VP...............T0170PMP061F07:............................F.....A....P...........T..E.......F...............L...........T.L........................L.....T0170PMP061C09:..........................................R...........D....................................................................T0170PMP124E06:...........A..............................R...........D..............................................................L.....T0170PMP111B02:......................................R...R...........D.......P............................................................T0170PMP062E08:...............D...........A...........V.............TD..................IT..........................................L.....T0170PMP062G03:......F........D...........A...........V.............TD..................IT..........................................L.....T0170PMP062G10:...............D...........A...........V...........A.TD..................IT..........................................L.....T0170PMP078E10:...............D...........A...........V.............TD..................VT..........................................L.....T0170PMP115E06:.......R.......D...........A...........V.............TD..................IT..........................................L.....T0170PMP122B02:...............D...........A...........V.............TD..A...............IT..........................................L.....T0170PMP061D07:...............D...........A..........RV.............TD..................IT................................................T0170PMP126D09:...............D...........A...........V.............TD..................IT................................................T0170PMP126B03:...............D.......................V.............TD...................T..........................................L.....T0170PMP126B02:K..............D.......................V.............TD...................T..........................................L.....T0170PMP075G11:...............D.......................V.............TD..................IT......H.........................................T0170PMP080E07:.........L.................A...........V.............TD..................IT................................................

TABLE A-2 Sequence alignment of CD3 cluster B binders T0170PMP060E11:EVQLVESGGGLVQPGGSLRLSCAASGDIYKSFDMGWYRQAPGKQRDLVAVIGSRGNNRGRTNYADSVKGRFTISRDGTGNTVYLLMNKLRPEDTAIYYCNTAPLVAGRPWGRGTLVTVSS

TABLE A-3 Sequence alignment of CD3 cluster C binders T0170PMP033G03:EVQLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADT0170PMP044A09:..........L............................S.....S................T0170PMP043E10:..........L............................S.....S....A...........T0170PMP044B10:..........L............................S.....S................T0170PMP033G03:SVKGRFTISRDNAKKTVYLQMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTQVTVSST0170PMP044A09:..............................................................T0170PMP043E10:..............................................................T0170PMP044B10:........................R...............................L.....

TABLE A-4 Sequence alignment of CD3 cluster D binders T0170PMP11A10:EVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTQVTVSS

TABLE A-5 Sequence alignment of CD3 cluster E binders T0170PMPO52G04:EVQLVESGGCAVQPGGSLRLSCAASGIRISRNMMGWFRQAPGKQRDLVARITPGGDTYYVDSVKGRFSISKDNAKNTVYLQMNSLKPEDTAVYYCNSYSTLGSWGQGTQVTVSST0170PMP062B02:......................V...............RT..RE..NM....S...A...............DS........D...............................T0170PMP114D01:......................V...............RT..RE..NM....S...A...............DS........D.........................L.....T0170PMP080F02:......................V...............RT..RG..NM....S...A...............DS........D...............................T0170PMP122A11:...........................................S...M....S................................R.....I................L.....T0170PMP113E06:......................V.......NH...........E...M....S......V.......................N..L...........R...............T0170PMP061E10:..........V...................THT.........E...EM..Q.S...K...I.......T.........F.R..D...D...............V....L.....T0170PMP061D03:..........V...................THT.........E...EM..Q.S...K...I.......T.........F.R..D...D...............V..........T0170PMP113E03:..........V...................THT.........E...EM..Q.S...K...I.......T.........F.R..D...DG..............V..........

TABLE A-6 Sequence alignment of CD3 cluster F binders T0170PMP05DA11:EVQLVESGGGLVQAGGSLRLSCAASGRSFNMNPLGWFRQSPGKEREFVAAHRWSDGNTYYVDSVKGRFTISRDNAKNTVYLQMNSLKSEDTAVYYCAAGRPWSAFRSPGEYVYWGQGTQVTVSST0170PMP044A06:...........................T.ST..M........N.................A.......A.......K..........................SY...D...............T0170PM5044D11:...........................A..T..M....FA...............................................................SY...D...............T0170PMP043E08:...........................A.S...M........R.........................................S..................SY...T.........L.....T0170PMP043E06:..........A..................ST..M....F.R...................A...............H...........................AH..N...............T0170PMP043E07:.............................SA..M....F......S..............A...............H...........................AH..N...............T0170PMP039D06:...........................G.ST..M..................................A..........H.......................DY.A.S..I............T0170PMP044F10:............R..............G.ST..M..................................A..........H.......................D..A.S..I.S.P........T0170PMP044B11:...........................A..T..M.............L..V..H..S...T...........................................YH..N.........L.....T0170PMP044C08:...........................A..T..M.............L..V..H..S...T..................C........................YH..N...............T0170PMP043B03:......F....................A.ST..M..............G.V.....S..SA........VF.................................YH..K.........L.....T0170PMP039E06:...............D...........A.ST..M......................S.FVA..................S........A...............YH..R..I...R........T0170PMP049A04:...............D...........A.ST..M......................S.FVA..................S........A...............YH..R..I...R..L.....T0170PMP043F09:...............A...........A.ST..M........Q....L..V..A......T......A........K.....L.N.....A.............YHA.L..I......L.....T0170PMP044C11:...............A...........A.ST..M........Q....L..V..A......T......A........K.....L.N.....A.............YHA.L..I............

TABLE A-7 Amino acid sequences of monovalent anti-CD3 Nanobodies (″ID″refers to the SEQ ID NO as used herein) Name Amino acid sequenceT0170PMP117G03  1EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP061F04  2EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMAWFRQSPGKEREFVAAIVWSOGNTYYEDFVKGRFTISRDSAKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP081D02  3EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMAWFRQSPGKEREFVAAIVWSDGNTYYEDFVKGRFTISRDSVKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP120D07  4EVOLVESGGGPVQAGGSLRLSCAASGRTYRGYSMAWFRQAPGKEREFVAAIVWSDGNTYYEDFVKGRFTISRDSAKNTLYLQMTNLEPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP118D11  5EVQLVESGGGPVQAGGSLRISCAASGRTYRGYSMAWFRQAPGKEREFVAAIVWSDGNTYYEDFVKGRFTISRDSAKNTYLQNITNLEPGDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP062A11  6EVQLVESGGGPVQAGGSLRLSCAASGRTVRGYSMAWFRQAPGKEREFVAAIVWSDGNTYYEDFVKGRFTSRDSAKNTISLHMTNLKPEDTAVYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP118A08  7EVQPVESGGGPVQAGGSLRLSCAASGRTYRGYSMAWFRQAPGKEREFVAAIVWSDGNTYYEDFVKGRFTISRDSAKNTLSLHMTNLKPEDTAVYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP122C07  8EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMAWFRQAPGKEREFVAAIVWSDGNTYYEDFVKGRFTISRDSAKNTSLHMANLKPEDTAVYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP062D09  9EVQLVESGGGPVQSGGSLRLSCANSGRTYRGYSMGWFRQAPGKEREFVAAIVWSDGNSYYEDFVKGRFTISRDSAKNIMYLQMTNLKPEDTAVYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP062G08 10EVQLVESGGGPVQAGGSLRLSCANSGRTYRGYSMAWFRQSPGKERGEVAAITWSEGNAYYEDFVKGRFTISRDNAKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP126E04 11EVQLVESGGGPVQAGGSLRLSCANSGRTYRGYSMAWFRQSPGKEREFVAAITWSEGNAYYEDIVKGRFTISRDNAKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP116E01 12EVQLVESGGGPVQAGGSLRLSCANSGRTYRGYSMAWFRQSPGKEREFVAAITWSEGNAYYEDFVRGRFTISRDNAKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP117E03 13EVQLVESGGGPVQAGGSLRLSCANSRRTYRGYSMAWFRQSPGKEREFVAAITWSEGNAYYEDFVKGRFTISRDNAKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP062B10 14EVQLVESGGGPVQAGGSLRLSCASSGRTYRGYSMAWFRQSPGKEREFVAAITWSEGNAYYEDFVKGRFTISRDNAKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP118E11 15EVQLVESGGGPVQAGGSLRLSCANSGRTYRGYSMAWFRQSPGKEREFVAAITWSEGNAYYEDFVKGRFTISRDNAKNTLYLQMTNLKPEDTALYYCAAKTRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP075A10 16EVQLVESGGGPVQAGGSLRLSCADSGRTYRGYSMAWFRQSPGKEREFVAAITWSEGNAYYEDFVKGRFTISRDNAKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTQWVSS T0170PMP062C06 17EVQLVESGGGPVQAGGSLRLSCANSGRTYRGYGMAWFRQSPGKEREFVAAITWSEGNAVEDFVKGRFTISRDNAKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP112G06 18EVQLVESGGGPVQAGGSLRLSCANSGRTYRGYSMAWFRQSPGKEREFVAAITWSEGNAYYEDFVKGRFTISRDNAKNTLYLQMTNLRPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP061A09 19EVQLVESGGGLVQAGGSLTLSCANSGRTYRGYSMAWFRQPPGKEREFVAAITWSEGNPYYEDFVKGRFTISRDNAKNTLYLRMTRLEPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTTVTVSS T0170PMP061B06 20EVQLVESGGGPVQAGGSLTLSCANSGRTYRGYSMAWFRQPPGKEREFVAAITWSEGNPYYEDFVKGRFTISRDNAKNTLYLRMTRLEPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP111C01 21EVQLVESGGGPVQAGGSLTLSCANSGRTYRGYSMAWFROPPGKEREFVAAITWSEGNPYYEDFAKGRFTISRDNAKNTLYLRMTRLEPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP061D06 22EVQLVESGGGPVQAGGSLTLSCANSGRAYRGYSMAWFRQPPGKEREFVAAITWSEGNPYYEDFVKGRFTISRDNAKNTLYLRMTRLEPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP061E09 23EVQLVESGGGPVQAGGSLILSCANSGRTYRGYSMAWFRQPPGKEREFVAAITWSEGNPYYEDFVKGRFTISRDNAKNTLYLRMTRLEPEDTALYYCAAKIRPYIFKVAGQYDYWGQGTQVTVSS T0170PMP078A07 24EVQLVESGGGPVQAGGSLTLSCANSRRTYRGYSMAWFRQPPGKEREFVAAITWSEGNPYYEDFVKGRFTSRDNAKNTLYLRMTRLEPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP061A04 25EVQLVESGGGPVQTGGSLRLSCVASGRTYRGYSMAWFRQPPGKEREFVAAITWSEGNTYYEDFVKGRFTISRDNAKNTLYLQMTGLKPEDTALYYCAAKIRPYIFKIPGQYDYWGQGTLVTVSS T0170PMP115A03 26EVQLVESGGGPVQTGGSLRLSCVASGRTYRGYSMAWFRQPPGKEREFVAAITWSEGNTYYEDFVKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCAAKIRPYIFKIPGQYDYWGQGTLVTVSS T0170PMP061F09 27EVQPVESGGGPVQTGGSLRLSCVASGRTYRGYSMAWFRQPPGKEREFVAAITWSEGNTYYEDFVKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCAAKIRPYIFKIPGQYDYWGQGTLVTVSS T0170PMP112D04 28EVQLVESGGGPVQTGGSLRLSCVASGRTYRGYSMAWFRQPPGKERGFVAAITWSEGNTYYEDFVKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCAAKIRPYIFKIPGQYDYWGQGTLVTVSS T0170PMP114E06 29EVQLVESGGGPVQTGGSLRLSCVAPGRTYRGYSMAWFRQPPGKEREFVAAITWSEGNTYYEDFVKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCAAKIRPYIFKIPGQYDYWGQGTLVTVSS T0170PMP113G04 30EVQLVESGGGPVQTGGSLRLSCVASGRTYRGYSMAWFRQPPGKEREFVAAITWSEGNTYYEDFVKGRFTISRDNAKNTLYLQMARKPEDTALYYCAAKIRPYIFKIPGQYDYWGQGTLVTVSS T0170PMP061D09 31EVQLVESGGGLVQTGGSLRLSCVASGRTYRGYSMAWFRQPPGKEREFVAAITWSEGNTYYEDFVKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCAAKIRPYIFKIPGQYDYWGQGTQVTVSS T0170PMP062G05 32EVQLVESGGGPVQTGGSLRLSCVASGRTYRGYSMAWFROPPGKEREFVAAITWSEGNTYYEDFVKGRFTISRDNAKSTLYLQMTSLKPEDTALWCAAKIRPYIFKIPGQYDYWGQGTQVTVSS T0170PMP114C05 33EVQLVESGGGPVQTGGSLRLSCVASGRTYRGYSMAWFRQPPGKEREFVAAITWSEGNTYYEDLVKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCAAKIRPYIFKIPGQYDYWGQGTQVTVSS T0170PMP117G05 34EVQLVESGGGPVQTGGSLRLSCVASGRTYRGYSMAWFRQPPGKEREFVAAITWSEGNTYYEDFVKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCAAKIRPYIFKVPGQYDYWGQGTQVTVSS T0170PMP061F07 35EVQLVESGGGPVQAGGSLRLSCAASGRTFRGYSMAWFRQPPGKEREFVAAITWSEGNTYYEDFVKGRFTSRDNAKNTLYLQMTSLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP061C09 36EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGREREFVAAIVWSDGNTYYEDSVKGRFTSRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP124E06 37EVQLVESGGGPAQAGGSLRLSCAASGRTYRGYSMGWFRQAPGREREFVAAIVWSDGNTYYEDSVKGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP111B02 38EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRRAPGREREFVAAIVWSDGNTYYEDPVKGRFTISRDNAKNTMYLQMTSLKPEDSAMCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP062E08 39EVQLVESGGGPVQAGDSLRLSCAASGRAYRGYSMGWFRQVPGKEREFVAAIVWTDGNTYYEDSVKGRFTISRDITKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP062G03 40EVQLVEFGGGPVQAGDSLRLSCAASGRAYRGYSMGWFRQVPGKEREFVAAIVWTDGNTYYEDSVKGRFTSRDITKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP062G10 41EVQLVESGGGPVQAGDSLRLSCAASGRAYRGYSMGWFRQVPGKEREFVAAIAWTDGNTYYEDSVKGRFTISRDITKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP078E10 42EVQLVESGGGPVQAGDSLRLSCAASGRAYRGYSMGWFRQVPGKEREFVAAIWTDGNTYYEDSVKGRFTSRDVTKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP115E06 43EVQLVESRGGPVQAGDSLRLSCAASGRAYRGYSMGWFRQVPGKEREFVAAIVWTDGNTYYEDSVKGRFTSRDITKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP122B02 44EVQLVESGGGPVQAGDSLRISCAASGRAYRGYSMGWFRQVPGKEREFVAAVWTDGNAYYEDSVKGRFTISRDITKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP061D07 45EVQLVESGGGPVQAGDSLRLSCAASGRAYRGYSMGWFRRVPGKEREFVAAIVWTDGNTYYEDSVKGRFTISRDITKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP126D09 46EVQLVSGGGPVQAGDSLRLSCAASGRAYRGYSMGWFRQVPGKEREFVAAIVWTDGNTYYEDSVKGRFTISRDITKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP126B03 47EVQLVESGGGPVQAGDSLRLSCAASGRTYRGYSMGWFRQVPGKEREFVAAIVWTDGNTYYEDSVKGRFTISRDNTKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP126B02 48KVQLVESGGGPVQAGDSLRLSCAASGRTYRGYSMGWFRQVPGKEREFVAAVWTDGNTYYEDSVKGRFTISRDNTKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS T0170PMP075G11 49EVQLVESGGGPVQAGDSLRLSCAASGRTYRGYSMGWFRQVPGKEREFVAAIVWTDGNTYYEDSVKGRFTSRDITKNTMYLHMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP080E07 50EVQLVESGGGLVQAGGSLRLSCAASGRAYRGYSMGWFRQVPGKEREFVAAIVWTDGNTYYEDSVKGRFTISRDITKNTYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSS T0170PMP060E11 51EVQLVESGGGLVQPGGSLRLSCAASGDIYKSFDMGWYRQAPGKQRDLVAVIGSRGNNRGRTNYADSVKGRFTSRDGTGNTVYLLMNKLRPEDTAWYCNTAPLVAGRPWGRGTLVTVSS T0170PMP011A10 52EVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFROPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTQVTVSS T0170PMP033G03 53EVQLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTATYYCAAGRPWSAYFISPAEYVHWGQGTQVTVSS T0170PMP044A09 54EVQLVESGGGLVQAGGSLRLSCAASGRTFSTNPMGWFRQSPGKERSIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTQVTVSS T0170PMP043E10 55EVQLVESGGGLVQAGGSLRISCAASGRTFSTNPMGWFRQSPGKERSLIAAARWADGNTFYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTQVTVSS T0170PMP044B10 56EVQLVESGGGLVQAGGSLRLSCAASGRTFSTNPMGWFRQSPGKERSLIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYLQMNSLRSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSS T0170PMP052G04 57EVQLVESGGGAVQPGGSLRLSCAASGIRISRNMMGWFRQAPGKQRDLVARITPGGDTYYVDSVKGRFSISKDNAKNTVYLQMNSLKPEDTAVYYCNSYSTLGSWGQGTQVTVSS T0170PMP062B02 58EVQLVESGGGAVQPGGSLRLSCVASGIRISRNMMGWFRRTPGRERNMVARISPGGATYYVDSVKGRFSISKDDSKNTVYLQMDSLKPEDTAVYYCNSYSTLGSWGQGTQVTVSS T0170PMP114D01 59EVQLVESGGGAVQPGGSLRLSCVASGIRISRNMMGWFRRTPGRERNMVARISPGGATYYVDSVKGRFSISKDDSKNTVYLQMDSLKPEDTAVYYCNSYSTLGSWGQGTLVTVSS T0170PMP080F02 60EVQLVESGGGAVQPGGSLRLSCVASGIRISRNMMGWFRRTPGRGRNVARISPGGATYYVDSVKGRFSISKDDSKNTVYLQMDSLKPEDTAVYYCNSYSTLGSWGQGTQVTVSS T0170PMP122A11 61EVQLVESGGGAVQPGGSLRLSCAASGIRISRNMMGWFRQAPGKSRDMVARISPGGDTYYVDSVKGRFSISKDNAKNTVYLQMNSLRPEDTAIYYCNSYSTLGSWGQGTTVTVSS T0170PMP113E06 62EVQLVESGGGAVQPGGSLRLSCVASGIRISNHMMGWFRQAPGEQRDMVARISPGGDTYVVDSVKGRFSISKDNAKNTVYLQMNNLKLEDTAVYYCNSYRTLGSWGQGTQVTVSS T0170PMP061E10 63EVQLVESGGGVVQPGGSLRLSCAASGIRISTHTMGWFRQAPEKQREMVAQISPGGKTYYIDSVKGRFTISKDNAKNTVFRMNDLKPDDTAVYYCNSYSTLGVWGQGTLVTVSS T0170PMP061D03 64EVQLVESGGGVVQPGGSLRLSCAASGIRISTHTMGWFRQAPEKQREMVAQISPGGKTYYIDSVKGRFTISKDNAKNTVFLRMNDLKPDDTAVYYCNSYSTLGVWGQGTQVTVSS T0170PMP113E03 65EVQLVESGGGVVQPGGSLRLSCAASGIRISTHTMGWFRQAPEKQREMVAQISPGGKTYYIDSVKGRFTISKDNAKNTVFLRMNDLKPDGTAVYYCNSYSTLGVWGQGTQVTVSS T0170PMP050A11 66EVQLVESGGGLVQAGGSLRLSCAASGRSFNMNPLGWFRQSPGKEREFVAAHRWSDGNTYYVDSVKGRFTISRDNAKNTVYLQMNSLKSEDTAVYYCAAGRPWSAFRSPGEYVYWGQGTQVTVSS T0170PMP044A06 67EVQLVESGGGLVQAGGSLRLSCAASGRTFSTNPMGWFRQSPGNEREFVAAHRWSDGNTYYADSVKGRFAISRDNAKKTVYLQMNSLKSEDTAVYYCAAGRPWSSYRSPDEYVYWGQGTQVTVSS T0170PMP044D11 68EVQLVESGGGLVQAGGSLRLSCAASGRAFNTNPMGWFRQFAGKEREFVAAHRWSDGNTYYVDSVKGRFTISRDNAKNTVYLQMNSLKSEDTAVYYCAAGRPWSSYRSPDEYVYWGQGTQVTVSS T0170PMP043E08 69EVQLVESGGGLVQAGGSLRLSCAASGRAFSMNPMGWFRQSPGREREFVAAHRWSDGNTYYVDSVKGRFTSRDNAKNTVYLQMSSLKSEDTAVYYCAAGRPWSSYRSPTEYVYWGQGTLVTVSS T0170PMP043E06 70EVQLVESGGGAVQAGGSLRLSCAASGRSFSTNPMGWFRQFPRKEREFVAAHRWSDGNTYYADSVKGRFTISRDNAKHTVYLQMNSLKSEDTAVYYCAAGRPWSAAHSPNEYVYWGQGTQVTVSS T0170PMP043E07 71EVQLVESGGGLVQAGGSLRLSCAASGRSFSANPMGWFRQFPGKERESVAAHRWSDGNTYYADSVKGRFTISRDNAKHTVYLQMNSLKSEDTAVYYCAAGRPWSAAHSPNEYVYWGQGTQVTVSS T0170PMP039D06 72EVQLVESGGGLVQAGGSLRLSCAASGRGISTNPMGWFRQSPGKEREFVAAHRWSDGNTYYVDSVKGRFAISRDNAKNTVHLQMNSLKSEDTAVYYCAAGRPWSDYRAPSEYIYWGQGTQVTVSS T0170PMP044F10 73EVQLVESGGGLVRAGGSLRLSCAASGRGFSTNPMGWFRQSPGKEREFVAAHRWSDGNTYYVDSVKGRFAISRDNAKNTVHLOMNSLKSEDTAVYYCAAGRPWSDFRAPSEYIYSGPGTQVTVSS T0170PMP044B11 74EVQLVESGGGLVQAGGSLRLSCAASGRAFNTNPMGWFRQSPGKEREFLAAVRWHDGSTYYTDSVKGRITISRDNAKNTVYLQMNSLKSEDTAVYYCAAGRPWSAYHSPNEYVYWGQGTLVTVSS T0170PMP044C08 75EVQLVESGGGLVQAGGSLRLSCAASGRAFNTNPMGWFRQSPGKEREFLAAVRWHDGSTYYTDSVKGRFTISRDNAKNTVCLQMNSLKSEDTAVYYCAAGRPWSAYHSPNEYVYWGQGTQVTVSS T0170PMP043B03 76EVQLVEFGGGLVQAGGSLRLSCAASGRAFSTNPMGWFRQSPGKEREFVGAVRWSDGSTYSADSVKGRTVFRDNAKNTVYLQMNSLKSEDTAVYYCAAGRPWSAYHSPKEYVYWGQGTLVTVSS T0170PMP039E06 77EVQLVESGGGLVQAGDSLRISCAASGRAFSTNPMGWFRQSPGKEREFVAAHRWSDGSTFVADSVKGRFTSRDNAKNTVSLQMNSLKSADTAVYYCAAGRPWSAYHSPREYIYWGRGTQVTVSS T0170PMP049A04 78EVQLVESGGGLVQAGDSLRLSCAASGRAFSTNPMGWFRQSPGKEREFVAAHRWSDGSTFVADSVKGRFTSRDNAKNTVSLQMNSLKSADTAVYYCAAGRPWSAYHSPREYIYWGRGTLVTVSS T0170PMP043F09 79EVQLVESGGGLVQAGASLRLSCAASGRAFSINPMGWFRQSPGQEREFLAAVRWADGNTYYTDSVKGRATSRDNAKKTVYLQMNLKSEDAAVYYCAAGRPWSAYHAPKEYSYWGQGTLVTVSS T0170PMP044C11 80EVQLVESGGGLVQAGASLRLSCAASGRAFSTNPMGWFRQSPGQEREFLAAVRWADGNTYYTDSVKGRATISRDNAKKTVYLQLNNLKSEDAAVYYCAAGRPWSAYHAPKEYIYWGQGTQVWSS

TABLE A-8Sequences for CDRs and frameworks, plus preferred combinations as provided in formula I,namely FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. ″ID″refers to the given SEQ ID NO. The first columnrefers to the SEQ ID NO of the complete ISV, i.e. FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. CDR1, CDR2and CDR3 were determined according to Kontermann, 2010. ID Name ID FR1ID CDR1 ID FR2 ID CDR2 ID FR3 ID CDR3 ID FR4  1 T0170PMP 144EVQLVESGGGPVQ  81 ERTYRGY 174 WFRQAPGK 101 AIVWSG 200YEDSVKGRFTISRDNAKNTM 123 KIRPYIFKAGQY 243 WGQGT 117G03 AGGSLRLSCAAS SMGEREFVA GNTY YLQMTSLKPEDSSTYYCAA DY QVTVSS  2 T0170PMP 144 EVQLVESGGGPVQ 82 GRTYRGY 175 WFRQSPGKE 102 AIVWSD 201 YEDFVKGRFTISRDSAKNTLY 123KIRPYIFKIAGQY 243 WGQGT 061F04 AGGSLRLSCAAS SMA REFVA GNTYLQMTNLKPEDTALYYCAA DY QVTVSS  3 T0170PMP 144 EVQLVESGGGPVQ  82 GRTYRGY175 FRQSPGKE 102 AIVWSD 202 YEDFVKGRFTISRDSVKNTLY 123 KIRPYIFKIAGQY 243WGQGT 081D02 AGGSLRLSCAAS SMA REFVA GNTY LQMTNLKPEDTALYYCAA DY QVTVSS  4T0170PMP 144 EVQLVESGGGPVQ  82 GRTYRGY 174 FRQAPGK 102 AIVWSD 203YEDFVKGRFTISRDSAKNTLY 123 KIRPYIFKIAGQY 243 WGQGT 120007 AGGSLRLSCAASSMA EREFVA GNTY LQMTNLEPEDTALYYCAA DY QVTVSS  5 T0170PMP 144EVQLVESGGGPVQ  82 GRTYRGY 174 FRQAPGK 102 AIVWSD 204EDFVKGRFTISRDSAKNTLY 123 KIRPYFKAGQY 244 WGQGT 118D11 AGGSLRLSCAAS SMAEREFVA GNTY LQMTNLEPGDTALYYCAA DY VTVSS  6  T0170PMP 144 EVQLVESGGGPVQ 82 GRTYRGY 174 FRQAPGK 102 AIVWSD 205 YEDFVKGRFTISRDSAKNTLS 123KIRPYFKAGQY 243 WGQGT 062A11 AGGSLRLSCAAS SMA EREFVA GNTYLHMTNLKPEDTAVYYCAA DY QVTVSS  7 T0170PMP 145 EVQPVESGGGPVQ  82 GRTYRGY174 FRQAPGK 102 AIVWSD 205 YEDFVKGRFTISRDSAKNTLS 123 KIRPYFKAGQY 243WGQGT 111A08 AGGSLRLSCAAS SMA EREFVA GNTY LHMTNLKPEDTAVYYCAA DY QVTVSS 8 T0170PMP 144 EVQLVESGGGPVQ  82 GRTYRGY 174 FRQAPGK 102 AIVWSD 206YEDFVKGRFTISRDSAKNTLS 123 KIRPYFKAGQY 244 WGQGT 122C07 AGGSLRLSCAAS SMAEREFVA GNTY LHMANLKPEDTAVYYCAA DY VTVSS  9 T0170PMP 146 EVQLVESGGGPVQ 81 ERTYRGY 174 WFRQAPGK 103 AIVWSD 207 YEDFVKGRFTISRDSAKNIM 123KIRPYFKAGQY 244 WGQGT 062D09 SGGSLRLSCANS SMG EREFVA GNSYYLQMTNLKPEDTAVYYCAA DY VTVSS 10 T0170PMP 147 EVQLVESGGGPVQ  82 GRTYRGY176 WFRQSPGKE 104 AITWSEG 208 YEDFVKGRFTISRDNAKNTL 123 KIRPYFKAGQY 244WGQGT 062G08 AGGSLRLSCANS SMA RGFVA NAY YLQMTNLKPEDTALYYCAA DY VTVSS 11T0170PMP 147 EVQLVESGGGPVQ  82 GRTYRGY 175 WFRQSPGKE 104 AITWSEG 208YEDFVKGRFTISRDNAKNTL 123 KIRPYFKAGQY 244 WGQGT 126E04 AGGSLRLSCANS SMAREFVA NAY YLQMTNLKPEDTALYYCAA DY VTVSS 12 T0170PMP 147 EVQLVESGGGPVQ  82GRTYRGY 175 WFRQSPGKE 104 AITWSEG 209 YEDFVRGRFTISRDNAKNTL 123KIRPYFKAGQY 244 WGQGT 116E01 AGGSLRLSCANS SMA REFVA NAYYLQMTNLKPEDTALYYCAA DY VTVSS 13 T0170PMP 147 EVQLVESGGGPVQ  83 RRTYRGY175 WFRQSPGKE 104 AITWSEG 208 YEDFVKGRFTISRDNAKNTL 123 KIRPYFKAGQY 244WGQGT 117E03 AGGSLRLSCANS SMA REFVA NAY YLQMTNLKPEDTALYYCAA DY VTVSS 14T0170PMP 148 EVQLVESGGGPVQ  82 GRTYRGY 175 WFRQSPGKE 104 AITWSEG 208YEDFVKGRFTISRDNAKNTL 123 KIRPYFKAGQY 244 WGQGT 062C10 AGGSLRLSCASS SMAREFVA NAY YLQMTNLKPEDTALYYCAA DY VTVSS 15 T0170PMP 147 EVQLVESGGGPVQ  82GRTYRGY 175 WFRQSPGKE 104 AITWSEG 208 YEDFVKGRFTISRDNAKNTL 124KTRPYIFKIAGQY 243 WGQGT 118E11 AGGSLRLSCANS SMA REFVA NAYYLQMTNLKPEDTALYYCAA DY QVTVSS 16 T0170PMP 149 EVQLVESGGGPVQ  82 GRTYRGY175 WFRQSPGKE 104 AITWSEG 208 YEDFVKGRFTISRDNAKNTL 123 KIRPYFKAGQY 243WGQGT 075A10 AGGSLRLSCADS SMA REFVA NAY YLQMTNLKPEDTALYYCAA DY QVTVSS 17T0170PMP 147 EVQLVESGGGPVQ  84 GRTYRGY 175 WFRQSPGKE 104 AITWSEG 208YEDFVKGRFTISRDNAKNTL 123 KIRPYFKAGQY 243 WGQGT 062C06 AGGSLRLSCANS GMAREFVA NAY YLQMTNLKPEDTALYYCAA DY QVTVSS 18 T0170PMP 147 EVQLVESGGGPVQ 82 GRTYRGY 175 WFRQSPGKE 104 AITWSEG 210 YEDFVKGRFTISRDNAKNTL 123KIRPYFKAGQY 243 WGQGT 112G06 AGGSLRLSCANS SMA REFVA NAYYLQMTNLRPEDTALYYCAA DY QVTVSS 19 T0170PMP 150 EVQLVESGGGLVQ  82 GRTYRGY177 WFRQPPGKE 105 AITWSED 211 YEDFVKGRFTISRDNAKNTL 123 KIRPYFKAGQY 244WGQGT 061A09 AGGSLTLSCANS SMA REFVA NPY YLRMTRLEPEDTALYYCAA DY VTVSS 20T0170PMP 151 EVQLVESGGGPVQ  82 GRTYRGY 177 WFRQPPGKE 105 AITWSED 211YEDFVKGRFTISRDNAKNTL 123 KIRPYFKAGQY 244 WGQGT 061B06 AGGSLTLSCANS SMAREFVA NPY YLRMTRLEPEDTALYYCAA DY VTVSS 21 T0170PMP 151 EVQLVESGGGPVQ  82GRTYRGY 177 WFRQPPGKE 105 AITWSED 212 YEDFAKGRFTISRDNAKNTL 123KIRPYFKAGQY 244 WGQGT 111C01 AGGSLTLSCANS SMA REFVA NPYYLRMTRLEPEDTALYYCAA DY VTVSS 22 T0170PMP 151 EVQLVESGGGPVQ  85 GRAYRGY177 WFRQPPGKE 105 AITWSED 211 YEDFVKGRFTISRDNAKNTL 123 KIRPYFKAGQY 243WGQGT 061D06 AGGSLTLSCANS SMA REFVA NPY YLRMTRLEPEDTALYYCAA DY QVTVSS 23T0170PMP 151 EVQLVESGGGPVQ  82 GRTYRGY 177 WFRQPPGKE 105 AITWSED 211YEDFVKGRFTISRDNAKNTL 125 KIRPYIFKVAGQ 243 WGQGT 061E09 AGGSLTLSCANS SMAREFVA NPY YLRMTRLEPEDTALYYCAA YDY QVTVSS 24 T0170PMP 151 EVQLVESGGGPVQ 83 RRTYRGY 177 WFRQPPGKE 105 AITWSED 211 YEDFVKGRFTISRDNAKNTL 123KIRPYFKAGQY 243 WGQGT 078A07 AGGSLTLSCANS SMA REFVA NPYYLRMTRLEPEDTALYYCAA DY QVTVSS 25 T0170PMP 152 EVQLVESGGGPVQ  82 GRTYRGY177 WFRQPPGKE 106 AITWSEG 213 YEDFVKGRFTISRDNAKNTL 126 KIRPYIFKIPGQY 244WGQGT 061A04 TGGSLRLSCVAS SMA REFVA NTY YLQMTGLKPEDTALYYCAA DY VTVSS 26T0170PMP 152 EVQLVESGGGPVQ  82 GRTYRGY 177 WFRQPPGKE 106 AITWSEG 214YEDFVKGRFTISRDNAKNTL 126 KIRPYIFKIPGQY 244 WGQGT 115A03 TGGSLRLSCVAS SMAREFVA NTY YLQMTSLKPEDTALYYCAA DY VTVSS 27 T0170PMP 153 EVQPVESGGGPVQ  82GRTYRGY 177 WFRQPPGKE 106 AITWSEG 214 YEDFVKGRFTISRDNAKNTL 126KIRPYIFKIPGQY 244 WGQGT 061F09 TGGSLRLSCVAS SMA REFVA NTYYLQMTSLKPEDTALYYCAA DY VTVSS 28 T0170PMP 152 EVQLVESGGGPVQ  82 GRTYRGY178 WFRQPPGKE 106 AITWSEG 214 YEDFVKGRFTISRDNAKNTL 126 KIRPYIFKIPGQY 244WGQGT 112D04 TGGSLRLSCVAS SMA RGFVA NTY YLQMTSLKPEDTALYYCAA DY VTVSS 29T0170PMP 154 EVQLVESGGGPVQ  82 GRTYRGY 177 WFRQPPGKE 106 AITWSEG 214YEDFVKGRFTISRDNAKNTL 126 KIRPYIFKIPGQY 244 WGQGT 114E06 TGGSLRLSCVAP SMAREFVA NTY YLQMTSLKPEDTALYYCAA DY VTVSS 30 T0170PMP 152 EVQLVESGGGPVQ  82GRTYRGY 177 WFRQPPGKE 106 AITWSEG 215 YEDFVKGRFTISRDNAKNTL 126KIRPYIFKIPGQY 244 WGQGT 113G04 TGGSLRLSCVAS SMA REFVA NTYYLQMASLKPEDTALYYCAA DY VTVSS 31 T0170PMP 155 EVQLVESGGGLVQ  82 GRTYRGY177 WFRQPPGKE 106 AITWSEG 214 YEDFVKGRFTISRDNAKNTL 126 KIRPYIFKIPGQY 243WGQGT 061D09 TGGSLRLSCVAS SMA REFVA NTY YLQMTSLKPEDTALYYCAA DY QVTVSS 32T0170PMP 152 EVQLVESGGGPVQ  82 GRTYRGY 177 WFRQPPGKE 106 AITWSEG 216YEDFVKGRFTISRDNAKSTLY 126 KIRPYIFKIPGQY 243 WGQGT 062G05 TGGSLRLSCVASSMA REFVA NTY LQMTSLKPEDTALYYCAA DY QVTVSS 33 T0170PMP 152 EVQLVESGGGPVQ 82 GRTYRGY 177 WFRQPPGKE 106 AITWSEG 217 YEDLVKGRFTISRDNAKNTLY 126KIRPYIFKIPGQY 243 WGQGT 114C05 TGGSLRLSCVAS SMA REFVA NTYLQMTSLKPEDTALYYCAA DY QVTVSS 34 T0170PMP 152 EVQLVESGGGPVQ  82 GRTYRGY177 WFRQPPGKE 106 AITWSEG 214 YEDFVKGRFTISRDNAKNTL 127 KIRPYIFKVPGQ 243WGQGT 117G05 TGGSLRLSCVAS SMA REFVA NTY YLQMTSLKPEDTALYYCAA YDY QVTVSS35 T0170PMP 144 EVQLVESGGGPVQ  86 GRTFRGY 177 WFRQPPGKE 106 AITWSEG 214YEDFVKGRFTISRDNAKNTL 123 KIRPYFKAGQY 244 WGQGT 061F07 AGGSLRLSCAAS SMAREFVA NTY YLQMTSLKPEDTALYYCAA DY VTVSS 36 T0170PMP 144 EVQLVESGGGPVQ  81ERTYRGY 179 WFRQAPGR 102 AIVWSD 200 YEDSVKGRFTISRDNAKNTM 123 KIRPYFKAGQY243 WGQGT 061C09 AGGSLRLSCAAS SMG EREFVA GNTY YLQMTSLKPEDSATYYCAA DYQVTVSS 37 T0170PMP 156 EVQLVESGGGPAQ  81 ERTYRGY 179 WFRQAPGR 102 AIVWSD200 YEDSVKGRFTISRDNAKNTM 123 KIRPYFKAGQY 244 WGQGT 124E09 AGGSLRLSCAASSMG EREFVA GNTY YLQMTSLKPEDSATYYCAA DY VTVSS 38 T0170PMP 144EVQLVESGGGPVQ  81 ERTYRGY 180 WFRRAPGRE 102 AIVWSD 218YEDPVKGRFTISRDNAKNT 123 KIRPYFKAGQY 243 WGQGT 111B02 AGGSLRLSCAAS SMGREFVA GNTY MYLQMTSLKPEDSATYYCA DY QVTVSS A 39 T0170PMP 157 EVQLVESGGGPVQ 87 GRAYRGY 181 WFRQVPGK 107 AIVWTD 219 YEDSVKGRFTISRDITKNTMY 123KIRPYFKAGQY 244 WGQGT 062E08 AGDSLRLSCAAS SMG EREFVA GNTYLQMTSLKPEDSATYYCAA DY VTVSS 40 T0170PMP 158 EVQLVEFGGGPVQ  87 GRAYRGY181 WFRQVPGK 107 AIVWTD 219 YEDSVKGRFTISRDITKNTMY 123 KIRPYFKAGQY 244WGQGT 062G03 AGDSLRLSCAAS SMG EREFVA GNTY LQMTSLKPEDSATYYCAA DY VTVSS 41T0170PMP 157 EVQLVESGGGPVQ  87 GRAYRGY 181 WFRQVPGK 108 AIAWTD 219YEDSVKGRFTISRDITKNTMY 123 KIRPYFKAGQY 244 WGQGT 062G10 AGDSLRLSCAAS SMGEREFVA GNTY LQMTSLKPEDSATYYCAA DY VTVSS 42 T0170PMP 157 EVQLVESGGGPVQ 87 GRAYRGY 181 WFRQVPGK 107 AIVWTD 220 YEDSVKGRFTISRDVTKNTM 123KIRPYFKAGQY 244 WGQGT 078E10 AGDSLRLSCAAS SMG EREFVA GNTYYLQMTSLKPEDSATYYCAA DY VTVSS 43 T0170PMP 159 EVQLVESRGGPVQ  87 GRAYRGY181 WFRQVPGK 107 AIVWTD 219 YEDSVKGRFTISRDITKNTMY 123 KIRPYFKAGQY 244WGQGT 115E09 AGDSLRLSCAAS SMG EREFVA GNTY LQMTSLKPEDSATYYCAA DY VTVSS 44T0170PMP 157 EVQLVESGGGPVQ  87 GRAYRGY 181 WFRQVPGK 109 AIVWTD 219YEDSVKGRFTISRDITKNTMY 123 KIRPYFKAGQY 244 WGQGT 122B02 AGDSLRLSCAAS SMGEREFVA GNAY LQMTSLKPEDSATYYCAA DY VTVSS 45 T0170PMP 157 EVQLVESGGGPVQ 87 GRAYRGY 182 WFRRVPGKE 107 AIVWTD 219 YEDSVKGRFTISRDITKNTMY 123KIRPYFKAGQY 243 WGQGT 061D07 AGDSLRLSCAAS SMG REFVA GNTYLQMTSLKPEDSATYYCAA DY QVTVSS 46 T0170PMP 160 EVQLVSGGGPVQA  87 GRAYRGY181 WFRQVPGK 107 AIVWTD 219 YEDSVKGRFTISRDITKNTMY 123 KIRPYFKAGQY 243WGQGT 126D09 GSLRLSCAAS SMG EREFVA GNTY LQMTSLKPEDSATYYCAA DY QVTVSS 47T0170PMP 157 EVQLVESGGGPVQ  81 ERTYRGY 181 WFRQVPGK 107 AIVWTD 221YEDSVKGRFTISRDNTKNTM 123 KIRPYFKAGQY 244 WGQGT 126B06 AGDSLRLSCAAS SMGEREFVA GNTY YLQMTSLKPEDSATYYCAA DY VTVSS 48 T0170PMP 161 KVQLVESGGGPVQ 81 ERTYRGY 181 WFRQVPGK 107 AIVWTD 221 YEDSVKGRFTISRDNTKNTM 123KIRPYFKAGQY 244 WGQGT 126B02 AGDSLRLSCAAS SMG EREFVA GNTYYLQMTSLKPEDSATYYCAA DY VTVSS 49 T0170PMP 157 EVQLVESGGGPVQ  81 ERTYRGY181 WFRQVPGK 107 AIVWTD 222 YEDSVKGRFTISRDITKNTMY 123 KIRPYFKAGQY 243WGQGT 075G11 AGDSLRLSCAAS SMG EREFVA GNTY LHMTSLKPEDSATYYCAA DY QVTVSS50 T0170PMP 162 EVQLVESGGGLVQ  87 GRAYRGY 181 WFRQVPGK 107 AIVWTD 219YEDSVKGRFTISRDITKNTMY 123 KIRPYFKAGQY 243 WGQGT 080E07 AGGSLRLSCAAS SMGEREFVA GNTY LQMTSLKPEDSATYYCAA DY QVTVSS 51 T0170PMP 163 EVQLVESGGGLVQ 88 GDIYKSF 183 WYRQAPGK 110 VIGSRGN 223 YADSVKGRFTISRDGTGNTV 128APLVAGRP 245 060E11 PGGSLRLSCAAS DMG QRDLVA NRGRTN YLLMNKLRPEDTAIYYCNT52 T0170PMP 164 EVQLVESGGGLVQ  89 GRTFSSY 177 WFRQPPGKE 111 SISWSGE 224YRNSVKGRFTISRDNAKNTV 129 KIAKTYPDNWY 243 WGQGT 011A10 GGGSLSLSCAAS AMAREFVA NTN YLQMNSLKPEDTAVYYCAA WTKSNNYNY QVTVSS 53 T0170PMP 165EVQLVESGGGSVQ  90 GRTFSTN 184 WFRQVPGK 112 AVRWAD 225YADSVKGRFTISRDNAKKTV 130 GRPWSAYHSPA 243 WGQGT 033G03 AGGSLRLSCAAS PMGERELIA GNTF YLQMNSLKSEDTATYYCAA EYVH QVTVSS 54 T0170PMP 162EVQLVESGGGLVQ  90 GRTFSTN 185 WFRQSPGKE 112 AVRWAD 225YADSVKGRFTISRDNAKKTV 130 GRPWSAYHSPA 243 WGQGT 044A09 AGGSLRLSCAAS PMGRSLIA GNTF YLQMNSLKSEDTATYYCAA EYVH QVTVSS 55 T0170PMP 162 EVQLVESGGGLVQ 90 GRTFSTN 185 WFRQSPGKE 113 AARWAD 225 YADSVKGRFTISRDNAKKTV 130GRPWSAYHSPA 243 WGQGT 043E10 AGGSLRLSCAAS PMG RSLIA GNTFYLQMNSLKSEDTATYYCAA EYVH QVTVSS 56 T0170PMP 162 EVQLVESGGGLVQ  90GRTFSTN 185 WFRQSPGKE 112 AVRWAD 226 YADSVKGRFTISRDNAKKTV 130GRPWSAYHSPA 244 WGQGT 044B10 AGGSLRLSCAAS PMG RSLIA GNTFYLQMNSLRSEDTATYYCAA EYVH VTVSS 57 T0170PMP 166 EVQLVESGGGAVQ  91 GIRISRN186 WFRQAPGK 114 RITPGGD 227 YVDSVKGRFSISKDNAKNTV 131 YSTLGS 243 WGQGT052G04 PGGSLRLSCAAS MMG QRDLVA TY YLQMNSLKPEDTAVYYCNS QVTVSS 58 T0170PMP167 EVQLVESGGGAVQ  91 GIRISRN 187 WFRRTPGRE 115 RISPGGA 228YVDSVKGRFSISKDDSKNTV 131 YSTLGS 243 WGQGT 062B02 PGGSLRLSCVAS MMG RNMVATY YLQMDSLKPEDTAVYYCNS QVTVSS 59 T0170PMP 167 EVQLVESGGGAVQ  91 GIRISRN187 WFRRTPGRE 115 RISPGGA 228 YVDSVKGRFSISKDDSKNTV 131 YSTLGS 244 WGQGT114D01 PGGSLRLSCVAS MMG RNMVA TY YLQMDSLKPEDTAVYYCNS VTVSS 60 T0170PMP167 EVQLVESGGGAVQ  91 GIRISRN 188 WFRRTPGRG 115 RISPGGA 228YVDSVKGRFSISKDDSKNTV 131 YSTLGS 243 WGQGT 080F02 PGGSLRLSCVAS MMG RNMVATY YLQMDSLKPEDTAVYYCNS QVTVSS 61 T0170PMP 166 EVQLVESGGGAVQ  91 GIRISRN189 WFRQAPGK 116 RISPGGD 229 YVDSVKGRFSISKDNAKNTV 131 YSTLGS 244 WGQGT122A11 PGGSLRLSCAAS MMG SRDMVA TY YLQMNSLRPEDTAIYYCNS VTVSS 62 T0170PMP167 EVQLVESGGGAVQ  92 GIRISNH 190 WFRQAPGE 116 RISPGGD 230VVDSVKGRFSISKDNAKNTV 132 YRTLGS 243 WGQGT 133E06 PGGSLRLSCVAS MMG QRDMVATY YLQMNNLKLEDTAVYYCNS QVTVSS 63 T0170PMP 168 EVQLVESGGGVVQ  93 GIRISTHT191 WFRQAPEK 117 QISPGGK 231 YIDSVKGRFTISKDNAKNTVF 133 YSTLGV 244 WGQGT061E10 PGGSLRLSCAAS MG QREMVA TY LRMNDLKPDDTAVYYCNS VTVSS 64 T0170PMP168 EVQLVESGGGVVQ  93 GIRISTHT 191 WFRQAPEK 117 QISPGGK 231YIDSVKGRFTISKDNAKNTVF 133 YSTLGV 243 WGQGT 061D03 PGGSLRLSCAAS MG QREMVATY LRMNDLKPDDTAVYYCNS QVTVSS 65 T0170PMP 168 EVQLVESGGGVVQ  93 GIRISTHT191 WFRQAPEK 117 QISPGGK 232 YIDSVKGRFTISKDNAKNTVF 133 YSTLGV 243 WGQGT113E03 PGGSLRLSCAAS MG QREMVA TY LRMNDLKPDGTAVYYCNS QVTVSS 66 T0170PMP162 EVQLVESGGGLVQ  94 GRSFNM 175 WFRQSPGKE 118 AHRWSD 233YVDSVKGRFTISRDNAKNTV 134 GRPWSAFRSPG 243 WGQGT 050A11 AGGSLRLSCAAS NPLGREFVA GNTY YLQMNSLKSEDTAVYYCAA EYVY QVTVSS 67 T0170PMP 162 EVQLVESGGGLVQ 90 GRTFSTN 192 WFRQSPGN 118 AHRWSD 234 YADSVKGRFAISRDNAKKTV 135GRPWSSYRSPD 243 WGQGT 044A06 AGGSLRLSCAAS PMG EREFVA GNTYYLQMNSLKSEDTAVYYCAA EYVY QVTVSS 68 T0170PMP 162 EVQLVESGGGLVQ  95 GRAFNT193 WFRQFAGKE 118 AHRWSD 233 YVDSVKGRFTISRDNAKNTV 135 GRPWSSYRSPD 243WGQGT 044D11 AGGSLRLSCAAS NPMG REFVA GNTY YLQMNSLKSEDTAVYYCAA EYVYQVTVSS 69 T0170PMP 162 EVQLVESGGGLVQ  96 GRAFSM 194 WFRQSPGRE 118 AHRWSD235 YVDSVKGRFTISRDNAKNTV 136 GRPWSSYRSPT 244 WGQGT 043E08 AGGSLRLSCAASNPMG REFVA GNTY YLQMSSLKSEDTAVYYCAA EYVY VTVSS 70 T0170PMP 169EVQLVESGGGAVQ  97 GRSFSTN 195 WFRQFPRKE 118 AHRWSD 236YADSVKGRFTISRDNAKHTV 137 GRPWSAAHSP 243 WGQGT 043E06 AGGSLRLSCAAS PMGREFVA GNTY YLQMNSLKSEDTAVYYCAA NEYVY QVTVSS 71 T0170PMP 162EVQLVESGGGLVQ  98 GRSFSAN 196 WFRQFPGKE 118 AHRWSD 236YADSVKGRFTISRDNAKHTV 137 GRPWSAAHSP 243 WGQGT 043E07 AGGSLRLSCAAS PMGRESVA GNTY YLQMNSLKSEDTAVYYCAA NEYVY QVTVSS 72 T0170PMP 162EVQLVESGGGLVQ  99 GRGFSTN 175 WFRQSPGKE 118 AHRWSD 237YVDSVKGRFAISRDNAKNTV 138 GRPWSDYRAPS 243 WGQGT 039D06 AGGSLRLSCAAS PMGREFVA GNTY HLQMNSLKSEDTAVYYCAA EYIY QVTVSS 73 T0170PMP 170 EVQLVESGGGLVR 99 GRGFSTN 175 WFRQSPGKE 118 AHRWSD 237 YVDSVKGRFAISRDNAKNTV 139GRPWSDFRAPS 246 SGPGTQ 044F10 AGGSLRLSCAAS PMG REFVA GNTYHLQMNSLKSEDTAVYYCAA EYIY VTVSS 74 T0170PMP 162 EVQLVESGGGLVQ  95 GRAFNT197 WFRQSPGKE 119 AVRWH 238 YTDSVKGRFTISRDNAKNTV 140 GRPWSAYHSPN 244WGQGT 044B11 AGGSLRLSCAAS NPMG REFLA DGSTY YLQMNSLKSEDTAVYYCAA EYVYVTVSS 75 T0170PMP 162 EVQLVESGGGLVQ  95 GRAFNT 197 WFRQSPGKE 119 AVRWH239 YTDSVKGRFTISRDNAKNTV 140 GRPWSAYHSPN 243 WGQGT 044C08 AGGSLRLSCAASNPMG REFLA DGSTY CLQMNSLKSEDTAVYYCAA EYVY QVTVSS 76 T0170PMP 171EVQLVEFGGGLVQ 100 GRAFSTN 198 WFRQSPGKE 120 AVRWSD 240SADSVKGRFTVFRDNAKNT 141 GRPWSAYHSPK 244 WGQGT 043B03 AGGSLRLSCAAS PMGREFVG GSTY VYLQMNSLKSEDTAVYYCA EYVY VTVSS A 77 T0170PMP 172EVQLVESGGGLVQ 100 GRAFSTN 175 WFRQSPGKE 121 AHRWSD 241VADSVKGRFTISRDNAKNTV 142 GRPWSAYHSPR 247 WGRGT 039E06 AGDSLRLSCAAS PMGREFVA GSTF SLQMNSLKSADTAVYYCAA EYIY QVTVSS 78 T0170PMP 172 EVQLVESGGGLVQ100 GRAFSTN 175 WFRQSPGKE 121 AHRWSD 241 VADSVKGRFTISRDNAKNTV 142GRPWSAYHSPR 245 WGRGTL 049A04 AGDSLRLSCAAS PMG REFVA GSTFSLQMNSLKSADTAVYYCAA EYIY VTVSS 79 T0170PMP 173 EVQLVESGGGLVQ 100 GRAFSTN199 WFRQSPGQ 122 AVRWAD 242 YTDSVKGRATISRDNAKKTV 143 GRPWSAYHAPK 244WGQGT 043F09 AGASLRLSCAAS PMG EREFLA GNTY YLQLNNLKSEDAAVYYCAA EYIY VTVSS80 T0170PMP 173 EVQLVESGGGLVQ 100 GRAFSTN 199 WFRQSPGQ 122 AVRWAD 242YTDSVKGRATISRDNAKKTV 143 GRPWSAYHAPK 243 WGQGT 044C11 AGASLRLSCAAS PMGEREFLA GNTY YLQLNNLKSEDAAVYYCAA EYIY QVTVSS

TABLE A-9Sequences of multispecific polypeptides (with and without tags). ″ID″refers to the SEQ ID NO as used herein. ID Name DescriptionAmino acid sequence 248 T017000001 T0170011A10-35GS-EVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLT0170011A10FLAG3-QMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGHIS6GSGGGGSEVQLVESGGGLVQGGGSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 249 T017000004 20CD019C07-5GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170033G03-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSEVQLVESGGGSVQAGGSLRLSCAASGRTFSTNPMFLAG3-HIS6GWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 250 T01700000520CD019C07-5GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170011A10-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAFLAG3-HIS6WFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 251 T01700000620CD019C07-5GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQcAbLys3-FLAG3-HIS6MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 252 T01700001020CD019C07-9GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170033G03-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGSVQAGGSLRLSCAASGRTFSTFLAG3-HIS6NPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 253 T01700001120CD019C07-9GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170011A10-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQGGGSLSLSCAASGRTFSSFLAG3-HIS6YAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 254 T01700001220CD019C07-9GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQcAbLys3-FLAG3-HIS6MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 255 T01700001620CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170033G03-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVFLAG3-HIS6QLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH256 T017000017 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170011A10-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVFLAG3-HIS6QLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 257 T017000018 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQcAbLys3-FLAG3-HIS6MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 258 T017000020 T0170033G03-9GS-EVQLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYL20CD019C07-QMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFLAG3-HIS6FSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 259 T017000021T0170033G03-EVQLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYL35GS-20CD019C07-QMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGFLAG3-HIS6GSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYKAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH260 T017000022 T0170011A10-35GSEVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYL20CD019C07-QMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGFLAG3-HIS6GSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 261 T017000023 cAbLys3(D1E)-35GSEVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVY20CD019C07-LLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGFLAG3-HIS6GGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 262 T017000024 T0170033G03-EVQLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYL35GS-cAbLys3-QMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGFLAG3-HIS6GSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 263 T017000026 T0170011A10-9GS-EVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVY20CD019C07-QMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCFLAG3-HIS6TFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 264 T017000027T0170011A10-35GS-EVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLcAbLys3-FLAG3-HIS6QMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 265 T017000034 T0170061F07-35GS-EVQLVESGGGPVQAGGSLRLSCAASGRTFRGYSMAWFRQPPGKEREFVAAITWSEGNTYYEDFVKGRFTISRDNAKNTLYL20CD019C07-QMTSLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEFLAG3-HIS6VQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH266 T017000036 T0170117G03-EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNTMY35GS-20CD019C07-LQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSFLAG3-HIS6EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH267 T017000039 T0170061F04-35GS-EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMAWFRQSPGKEREFVAAIVWSDGNTYYEDFVKGRFTISRDSAKNTLYL20CD019C07-QMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEFLAG3-HIS6VQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAWYCAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH268 T017000040 T0170060E11-35GS-EVQLVESGGGLVQPGGSLRLSCAASGDIYKSFDMGWYRQAPGKQRDLVAVIGSRGNNRGRTNYADSVKGRFTISRDGTGN20CD019C07-TVYLLMNKLRPEDTAIYYCNTAPLVAGRPWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQFLAG3-HIS6LVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVMSNSLKDRFSESEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVYVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH269 T017000043 T0170050A11-35GS-EVQLVESGGGLVQAGGSLRLSCAASGRSFNMNPLGWFRQSPGKEREFVAAHRWSDGNTYYVDSVKGRFTISRDNAKNTVY20CD019C07-LQMNSLKSEDTAVYYCAAGRPWSAFRSPGEYVYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGFLAG3-HIS6GSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH270 T017000045 T0170033G03-EVQLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYL35GS-20CD019C07-QMNSLKSEDTATYYCAAGRPWSAYFISPAEYVHWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGFLAG3-HIS6GSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH271 T017000047 T0170011A10-35GSEVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYL20CD019C07-QMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGFLAG3-HIS6GSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 272 T017000048 T0170052G04-EVQLVESGGGAVQPGGSLRLSCAASGIRISRNMMGWFRQAPGKQRDLVARITPGGDTYYVDSVKGRFSISKDNAKNTVYLQ35GS-20CD019C07-MNSLKPEDTAVYYCNSYSTLGSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGFLAG3-HIS6LVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 273T017000052 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170061F04-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVFLAG3-HIS6QLVESGGGPVQAGGSLRLSCAASGRTYRGYSMAWFRQSPGKEREFVAAIVWSDGNTYYEDFVKGRFTISRDSAKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH274 T017000053 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170060E11-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVFLAG3-HIS6QLVESGGGLVQPGGSLRLSCAASGDIYKSFDMGWYRQAPGKQRDLVAVIGSRGNNRGRTNYADSVKGRFTISRDGTGNTVYLLMNKLRPEDTAIYYCNTAPLVAGRPWGRGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH275 T017000056 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVMSNSLKDRFSISEDSVKNAVYLQT0170052G04-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVFLAG3-HIS6QLVESGGGAVQPGGSLRLSCAASGIRISRNMMGWFRQAPGKQRDLVARITPGGDTYYVDSVKGRFSISKDNAKNTVYLQMNSLKPEDTAVYYCNSYSTLGSWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 276T017000057 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170050A11-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVFLAG3-HIS6QLVESGGGLVQAGGSLRLSCAASGRSFNMNPLGWFRQSPGKEREFVAAHRWSDGNTYYVDSVKGRITISRDNAKNTVYLQMNSLKSEDTAVYYCAAGRPWSAFRSPGEYVYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH277 T017000059 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170033G03-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVFLAG3-HIS6QLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH278 T017000061 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170011A10-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVFLAG3-HIS6QLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 279 T017000062 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170117G03-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVFLAG3-HIS6QLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH280 T017000066 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170061D09-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVFLAG3-HIS6QLVESGGGLVQTGGSLRLSCVASGRTYRGYSMAWFROPPGKEREFVAAITWSEGNTYYEDFVKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCAAKIRPYIFKIPGQYDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH281 T017000071 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170061F07-MNSLKPEDTAVYYCAAVRQMYM1VVPDYINGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVFLAG3-HIS6QLVESGGGPVQAGGSLRLSCAASGRTFRGYSMAWFRQPPGKEREFVAAITWSEGNTYYEDFVKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCAAKIRPY1FKIAGQYDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH282 T017000072 T0170061D09-35GS-EVQLVESGGGLVQTGGSLRLSCVASGRTYRGYSMAWFRQPPGKEREFVAAITWSEGNMEDFVKGRFTISRDNAKNTLYLQ20CD019C07-MTSLKPEDTALYYCAAKIRPYIFKIPGQYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVFLAG3-HIS6QLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVMSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH283 T017000084 20CD019C07(E1D)-DVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQ35GS-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVT0170117G03-AQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNIMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSA 284 T017000090RSV007B02-35GS-EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLT0170061F04-QMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGFLAG3-HIS6GGSEVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMAWFRQSPGKEREFVAAIVWSDGNTYYEDFVKGRFTISRDSAKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH285 T017000091 RSV007B02-35GS-EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLT0170117G03-QMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGFLAG3-HIS6GGSEVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRITISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 286 T017000094 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFROAPGKEREFVAEVRWGGVMSNSLKDRFSISEDSVKNAVYLQT0170117G03-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEV35GS-AL811-FLAG3-QLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNTMYLQHIS6MTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTESSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 287T017000096 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLT0170060E11-35GS-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVAL811-FLAG3-HIS6QLVESGGGLVQPGGSLRLSCAASGDIYKSFDMGWYRQAPGKQRQLVAVIGSRGNNRGRTNYADSVKGRFTISRDGTGNTVYLLMNKLRPEDTAIYYCNTAPLVAGRPWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTrLYLQMNSIRPEDTAVYYCTIGGSISRSSQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 288T017000100 T0170117G03-EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNMEDSVKGRFTISRDNAKNTY35GS-LQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSHER2005F07(Q108L)-EVQLVESGGGLVQAGGSLRLSCAASGFTFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMFLAG3-HIS6NSLKPEDTAVYYCKRFRTAAQGTDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH289 T017000101 HER200SF07(Q108L)-EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQM35GS-NSLKPEDTAVYYCKRFRTAAQGTDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVEST0170117G03-GGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNTMYLQMTSLKFLAG3-HIS6PEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH290 T017000088 RSV007B02(E1D)-DVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVY35GS-T0170055A02-QMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGAGGSEVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSSA 305 T017000001 T0170011A10EVQLVESGGGLVQGGGSLASCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYQMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSS 306 T01700000420CD019C07-5GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0174033G03MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSEVQLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSS 307 T017000005 20CD019C07-5GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVMSNSLKDRFSISEDSVKNAVYLQT0170011A10MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSS 308 T017000006 20CD019C07-5GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQcAbLys3MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSS 309 T017000010 20CD019C07-9GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170033G03MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKWYLQMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSS 310 T017000011 20CD019C07-9GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170011A10MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSS 311 T017000012 20CD019C07-9GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQcAbLys3MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSS 312 T017000016 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVMSNSLKDRFSISEDSVKNAVYLQT0170033G03MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSS 313 T01700001720CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170011A10MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSS 314 T01700001820CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQcAbLys3MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSS 315 T017000020T0170033G03-9GS-EVQLVESGGGSVQAGGSLRLSCAASGRTTSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYL20CD019C07QMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 316 T017000021 T0170033G03-EVQLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYL35GS-20CD019C07QMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 317 T017000022T0170011A10-35GS-EVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYL20CD019C07QMNSLSPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 318 T017000023cAbLys3(D1E)-35GS-EVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVY20CD019C07LLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 319 T017000024T0170033G03-EVQLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYL35GS-cAbLys3QMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSS 320 T017000026T0170011A10-9GS-EVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYL20CD019C07QMNSLKPEDTAVYYCAAKIAKTYPDNWYWTIGNNYNYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVMSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 321 T017000027 T0170011A10-35GS-EVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLcAbLys3QMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSS 322T017000034 T0170061F07-35GS-EVQLVESGGGPVQAGGSLRLSCAASGRTFRGYSMAWFRQPPGKEREFVAAITWSEGNTYYEDFVKGRFTISRDNAKNTLYL20CD019C07QMTSLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYKAAVRQMYMTVVPDYWGQGTLVTVSS 323 T017000036 T0170117G03-EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNTMY35GS-20CD019C07LQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVMSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 324 T017000039 T0170061F04-35GS-EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMAWFRQSPGKEREFVAAIVWSDGNTYYEDFVKGRFTISRDSAKNTLYL20CD019C07QMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTSGGTESSYTMGWFRQAPGKEREFVAEVRWGGVMSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 325 T017140040 T0170060E11-35GS-EVQLVESGGGLVQPGGSLRLSCAASGDIYKSFDMGWYRQAPGKQRDLVAVIGSRGNNRGRTNYADSVKGRFTISRDGTGN20CD019C07TVYLLMNKLRPEDTAIYYCNTAPLVAGRPWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMNVPDYWGQGTLVTVSS 326 T017000043 T0170050A11-35GS-EVQLVESGGGLVQAGGSLRLSCAASGRSFNMNPLGWFRQSPGKEREFVAAHRWSDGNTYYVDSVKGRFTISRDNAKNTVY20CD019C07LQMNSLKSEDTAVYYCAAGRPWSAFRSPGEYVYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 327 T017000045 T0170033G03-EVQLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYL35GS-20CD019C07QMNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVMSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 328 T017000047T0170011A10-35GS-EVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYL20CD019C07QMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 329 T017000048T0170052G04-EVQLVESGGGAVQPGGSLRLSCAASGIRISRNMMGWFRQAPGKQRDLVARITPGGDTYYVDSVKGRFSISKDNAKNTVYLQ35GS-20CD019C07MNSLKPEDTAVYYCNSYSTLGSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 330 T017000052 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170061F04MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMAWFRQSPGKEREFVAAIVWSDGNTYYEDFVKGRFTISRDSAKNTLYLQMTNLKPEDTALYKAAKIRPYIFKIAGQYDYWGQGTLVTVSS 331 T017000053 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170060E11MNSLSPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGDIYKSFDMGWYRQAPGKQRDLVAVIGSRGNNRGRTNYADSVKGRFTISRDGTGNWYLLMNKLRPEDTAIYYCNTAPLVAGRPWGRGTLVTVSS 332 T017000056 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170052G04MNSLKPEDTAVYKAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGAVQPGGSLRLSCAASGIRISRNMMGWFRQAPGKQRDLVARITPGGDTYYVDSVKGRFSISKDNAKNTVYLOMNSLKPEDTAVYYCNSYSTLGSWGQGTLVTVSS 333 T017000057 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170050A11MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRSFNMNPLGWFRQSPGKEREFVAAHRWSDGNTYYVDSVKGRFTISRDNAKNTVYLOMNSLKSEDTAWYCAAGRPWSAFRSPGEYVYWGQGTLVTVSS 334 T01700005920CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170033G03MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLMSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGGSLRLSCAASGRTFSTNPMGWFRQVPGKERELIAAVRWADGNTFYADSVKGRFTISRDNAKKTVYLQNSLKSEDTATYYCAAGRPWSAYHSPAEYVHWGQGTLVTVSS 335 T01700006120CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVMSNSLKDRFSISEDSVKNAVYLQT0170011A10MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKIAKTYPDNWYWTKSNNYNYWGQGTLVTVSS 336 T01700006220CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170117G03MNSLSPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNIMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS 337 T01700006620CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170061D09MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGGSLRLSCVASGRTYRGYSMAWFRQPPGKEREFVAAITWSEGNTYYEDFVKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCAAKIRPYIFKIPGQYDYWGQGTLVTVSS 338 T01700007120CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170061F07MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGPVQAGGSLRLSCAASGRTFRGYSMAWFRQPPGKEREFVAAITINSEGNTYYEDFVKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS 339 T017000072T0170061D09-35GS-EVQLVESGGGLVQTGGSLRLSCVASGRTYRGYSMAWFRQPPGKEREFVAAITWSEGNMEDFVKGRFTISRDNAKNTLYLQ20CD019C07MTSLKPEDTALYYCAAKIRPYWKIPGQYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLSPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 340 T017000084 20CD019C07(E1D)-DVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQ35GS-T0170117G03MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS 341 T017000090RSV007B02-35GS-EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLT0170061F04QMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMAWFRQSPGKEREFVAAIVWSDGNTYYEDFVKGRFTISRDSAKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS 342 T017000091RSV007B02-35GS-EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYLT0170117G03QMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSS 343 T01700009420CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170117G03-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEV35GS-ALB11QLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTFLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 344 T017000096 20CD019C07-35GS-EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQT0170060E11-35GS-MNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVALB11QLVESGGGLVQPGGSLRLSCAASGDIYKSFDMGWYRQAPGKQRDLVAVIGSRGNNRGRTNYADSVKGRFTISRDGTGNWYLLMNKLRPEDTAIYYCNTAPLVAGRPWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 345 T017000100 T0170117G03-EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNTMY35GS-LQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSHER2005F07(Q108L)EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTLVTVSS 346 T017000101 HER2005F07(Q108L)-EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQM35GS-NSLKPEDTAVYYCKRFRTAAQGTDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVEST0170117G03GGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVWSS 347 T017000088 RSV007802(E1D)-DVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNTVYL35GS-T0170055A02QMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHMGDATMADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLVTVSS

TABLE A-10 Sequences of components of TCR complex. ″ID″refers to the SEQ ID NO as used herein ID Name Amino acid sequence 291HUMAN CD3 DELTAMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMC(P04234)QSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK292 HUMAN CD3 GAMMAMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKG(P09693)SQNKSKPLQVYYRMCQNCIELAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRNAATISGFLFRN 293 HUMAN CD3 EPSILONMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFS(P07766)ELEQSGYYVCYPPPVPNPDYEPIRKGQRDLYSGLNQRRIRGSKPEDANFNCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPYLYLRARVCE 294 HUMAN CD3 ZETAMKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR(P20963)DPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR295 HUMAN TCR ALPHAPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFCONSTANT DOMAIN PSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(P01848) 296 HUMAN TCR BETAEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNCONSTANT DOMAINPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(P01850) 379 HUMAN CD3 GAMMAMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKG(P09693)SQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN  80 HUMAN CD3 EPSILONMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFS(P07766)ELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI 381 HUMAN TCR ALPHAIQVEQSPPDLILQEGANSTLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATTVATERYSLLYISSSQTTDSGVYFCAALIQVARIABLE DOMAIN GAQKLVFGQGTRLTIN DERIVED FROM 2IAN 382 HUMAN TCR BETANAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELAAPSQTSVVARIABLE DOMAIN YFCASTYHGTGYFGEGSWLTVV DERIVED FROM 2IAN 383HUMAN TCR ALPHAQLLEQSPQFLSIQEGENLTVYCNSSSVFSSLQWYRQEPGEGPVLLVTVVTGGEVKKLKRLTFQFGDARKDSSLHITAAQPGDTGLYLCAGAVARIABLE DOMAIN GSQGNLIFGKGTKLSVK DERIVED FROM 2XN9  84 HUMA TCR BETADGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLVARIABLE DOMAIN CASSSRSSYEQYFGPGTRLTVT DERIVED FROM 2XN9 385HUMAN TCR ALPHAGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCTVARIABLE DOMAIN VYGGATNKLIFGTGTLLAVQ DERIVED FROM 3TOE 386HUMAN TCR BETAVVSQHPSWVIAKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYIVARIABLE DOMAIN CSARGGSYNSPLHFGNGTRLTVT DERIVED FROM 3TOE

TABLE A-11 Sequences of TAA building blocks. ″ID″refers to the SEQ ID NO as used herein ID Name Amino acid sequence 297HER2005F07EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYQMNSLKPEDT(Q108L) AVYYCKRFRTAAQGTDYWGQGTLVTVSS 298 HER2047D05EVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVAISRVGVTSSADSVKGRFTISRVNAKDTVYLQMNSLKPED(L108Q) TAVYYCYMDQRLDGSTLAYWGQGTQVTVSS 299 EGFR009G08EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSS 300 bCEA5EVQLVESGGGSVQAGGSLRLSCAASGDTYGSYWMGWFRQAPGKEREGVAAINRGGGYTVYADSVKGRFTISRDTAKNTVYLQMNSLR(CEA#1) PDDTADYYCAASGVLGGLHEDWFNYWGQGTLVTVSS 301 T023200005EVQLVESGGGSVQAGGSLRLSCAASGDTYGSYWMGWFRQAPGQEREAVAAINRGGGYTVYADSVKGRFTISRDNAKNTYLQMNSL(CEA#5) RPDDTADYYCAASGVLGGLHEDWFNYWGQGTLVTVSS 302 7D12EVQLVESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGSWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSK(EGFR#1) PEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTLVTVSS 303 T02320003EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGSWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSK(EGFR#33) PEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSS 304 20CD019C07EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 387 cAblys3DVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSS 388 RSV007B02EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRDNAKNWYLQMNSLKPEDTAVYYCAADLTSTNPGSYIYIWAYDYWGQGTQVTVSS

1. Polypeptide comprising a first and a second immunoglobulin singlevariable domain (ISV), wherein said first ISV has high affinityfor/binds to cluster of differentiation 3 (CD3) present on a T cell;said second ISV has high affinity for/binds to a first antigen on atarget cell; wherein said first antigen is different from said CD3; andwherein said target cell is different from said T cell. 2.-12.(canceled)
 13. The polypeptide according to claim 1, wherein said firstISV essentially consists of 4 framework regions (FR1 to FR4,respectively) and 3 complementarity determining regions (CDR1 to CDR3,respectively), in which: (i) CDR1 is chosen from the group consistingof: (a) SEQ ID NOs: 81-100; and (b) amino acid sequences that have 4, 3,2, or 1 amino acid(s) difference with the amino acid sequence of SEQ IDNO: 81 or with any of SEQ ID NOs: 81-100; and/or (ii) CDR2 is chosenfrom the group consisting of: (c) SEQ ID NOs: 101-122; and (d) aminoacid sequences that have 4, 3, 2, or 1 amino acid(s) difference with theamino acid sequence of SEQ ID NO: 101 or with any of SEQ ID NOs:101-122; and/or (iii) CDR3 is chosen from the group consisting of: (e)SEQ ID NOs: 123-143; and (f) amino acid sequences that have 3, 2, or 1amino acid(s) difference with the amino acid sequence of SEQ ID NO: 123or with any of SEQ ID NOs: 123-143. 14.-43. (canceled)
 44. Thepolypeptide according to claim 1, wherein said first antigen on a targetcell is a tumour antigen, preferably a tumour associated antigen (TAA).45. The polypeptide according to claim 1, further comprising a thirdISV, which has high affinity for/binds to a second antigen on a targetcell, wherein said second antigen is different from said first antigen.46. The polypeptide according to claim 45, wherein said second antigenon a target cell is a tumour antigen, preferably a tumour associatedantigen (TAA). 47.-51. (canceled)
 52. The polypeptide according to claim1, further comprising a serum protein binding moiety.
 53. (canceled) 54.The polypeptide according to claim 52, wherein said serum proteinbinding moiety is an ISV binding serum albumin. 55.-64. (canceled)
 65. Apolypeptide that specifically binds CD3 and that comprises oressentially consists of 4 framework regions (FR1 to FR4, respectively)and 3 complementarity determining regions (CDR1 to CDR3, respectively),in which: (i) CDR1 is chosen from the group consisting of: (a) SEQ IDNOs: 81-100; or (b) amino acid sequences that have 4, 3, 2, or 1 aminoacid(s) difference with the amino acid sequence of any of SEQ ID NOs:81-100, provided that the polypeptide comprising the CDR1 with 4, 3, 2,or 1 amino acid(s) difference binds CD3 with about the same or a higheraffinity compared to the binding by the polypeptide comprising the CDR1without the 4, 3, 2, or 1 amino acid(s) difference, said affinity asmeasured by surface plasmon resonance; and/or (ii) CDR2 is chosen fromthe group consisting of: (c) SEQ ID NOs: 101-122; or (d) amino acidsequences that have 4, 3, 2, or 1 amino acid(s) difference with theamino acid sequence of any of SEQ ID NOs: 101-122, provided that thepolypeptide comprising the CDR2 with 4, 3, 2, or 1 amino acid(s)difference binds CD3 with about the same or a higher affinity comparedto the binding by the polypeptide comprising the CDR2 without the 4, 3,2, or 1 amino acid(s) difference, said affinity as measured by surfaceplasmon resonance; and/or (iii) CDR3 is chosen from the group consistingof: (e) SEQ ID NOs: 123-143; or (f) amino acid sequences that have 4, 3,2, or 1 amino acid(s) difference with the amino acid sequence of any ofSEQ ID NOs: 123-143, provided that the polypeptide comprising the CDR3with 4, 3, 2, or 1 amino acid(s) difference binds CD3 with about thesame or a higher affinity compared to the binding by the polypeptidecomprising the CDR3 without the 4, 3, 2, or 1 amino acid(s) difference,said affinity as measured by surface plasmon resonance. 66.-96.(canceled)
 97. The polypeptide according to claim 65, further comprisinga serum protein binding moiety.
 98. (canceled)
 99. The polypeptideaccording to claim 97, wherein said serum protein binding moiety is anISV that binds serum albumin. 100.-104. (canceled)
 105. A nucleic acidor nucleic acid sequence encoding a polypeptide as defined in claim 1,or a vector comprising said nucleic acid or nucleic acid sequence. 106.(canceled)
 107. A host cell transformed or transfected with the nucleicacid nucleic acid sequence, or vector as defined in claim
 105. 108. Aprocess for the production of a polypeptide, said process comprisingculturing a host cell as defined in claim 107 under conditions allowingthe expression of the polypeptide and recovering the producedpolypeptide from the culture.
 109. A pharmaceutical compositioncomprising a polypeptide according to claim
 1. 110. A method fortreating a subject in need thereof comprising administering to a subjectin need thereof the polypeptide according to claim
 1. 111. (canceled)112. (canceled)
 113. A method for the treatment or amelioration of adisease selected from the group consisting of a proliferative disease,an inflammatory disease, an infectious disease and an autoimmunedisease, comprising the step of administering to a subject in needthereof the polypeptide according to claim
 1. 114. The method accordingto claim 113, wherein said proliferative disease is cancer.
 115. Themethod according to claim 114, wherein said cancer is chosen from thegroup consisting of carcinomas, gliomas, mesotheliomas, melanomas,lymphomas, leukemias, adenocarcinomas: breast cancer, ovarian cancer,cervical cancer, glioblastoma, multiple myeloma (including monoclonalgammopathy of undetermined significance, asymptomatic and symptomaticmyeloma), prostate cancer, and Burkitt's lymphoma, head and neck cancer,colon cancer, colorectal cancer, non-small cell lung cancer, small celllung cancer, cancer of the esophagus, stomach cancer, pancreatic cancer,hepatobiliary cancer, cancer of the gallbladder, cancer of the smallintestine, rectal cancer, kidney cancer, bladder cancer, prostatecancer, penile cancer, urethral cancer, testicular cancer, vaginalcancer, uterine cancer, thyroid cancer, parathyroid cancer, adrenalcancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skincancer, retinoblastomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma,Kaposi's sarcoma, multicentric Castleman's disease or AIDS-associatedprimary effusion lymphoma, neuroectodermal tumors, rhabdomyosarcoma; aswell as any metastasis of any of the above cancers, as well asnon-cancer indications such as nasal polyposis. 116.-117. (canceled)